U.S. patent number 7,076,372 [Application Number 10/757,963] was granted by the patent office on 2006-07-11 for crystal structure of mvas.
This patent grant is currently assigned to Takeda San Diego, Inc.. Invention is credited to Alexei Brooun, David J. Hosfield, Robert J. Skene, Leslie W. Tari, Sheng Ye.
United States Patent |
7,076,372 |
Brooun , et al. |
July 11, 2006 |
Crystal structure of MvaS
Abstract
Provided are structure coordinates relating to MvaS and its
various uses.
Inventors: |
Brooun; Alexei (San Diego,
CA), Hosfield; David J. (San Diego, CA), Skene; Robert
J. (San Diego, CA), Tari; Leslie W. (San Diego, CA),
Ye; Sheng (Allen, TX) |
Assignee: |
Takeda San Diego, Inc. (San
Diego, CA)
|
Family
ID: |
36644182 |
Appl.
No.: |
10/757,963 |
Filed: |
January 14, 2004 |
Current U.S.
Class: |
702/19;
703/11 |
Current CPC
Class: |
C07K
1/306 (20130101); C12N 9/88 (20130101); C40B
30/04 (20130101); C07K 2299/00 (20130101) |
Current International
Class: |
G01N
33/48 (20060101) |
Field of
Search: |
;702/19 ;703/11 |
Primary Examiner: Marschel; Ardin H.
Assistant Examiner: Skibinsky; Anna
Attorney, Agent or Firm: Weitz; David J.
Claims
We claim:
1. A method for displaying a three dimensional representation of a
structure of a protein comprising: taking machine readable data
comprising structure coordinates that have a root mean square
deviation equal to or less than 0.98 Angstrom when compared to the
structure coordinates of FIG. 3, the root mean square deviation
being calculated such that the alpha-carbon atom positions of each
set of structure coordinates are superimposed and the root mean
square deviation is based only on those amino acid residues in the
structure coordinates that are also present in the portion of the
protein specified in Table 2; computing a three dimensional
representation of a structure based on the structure coordinates;
and displaying the three dimensional representation.
2. A method according to claim 1, wherein the root mean square
deviation is less than or equal to 0.65 Angstrom.
3. A method according to claim 1, wherein the root mean square
deviation is less than or equal to 0.49 Angstrom.
4. A method according to claim 1, wherein the root mean square
deviation comparison is also based on main-chain atoms positions of
the amino acid residues and the root mean square deviation is equal
to or less than 0.98 Angstrom.
5. A method according to claim 1, wherein the root mean square
deviation comparison is also based on non-hydrogen atoms positions
of the amino acid residues and the root mean square deviation is
equal to or less than 0.89 Angstrom.
Description
FIELD OF THE INVENTION
The present invention relates to a mevalonate pathway enzyme
responsible for the synthesis of 3-Hydroxy-3-methylglutaryl-CoA
(HMG-CoA) and more specifically to MvaS also known as HMG-CoA
Synthase. Provided is MvaS in crystalline form, methods of forming
crystals comprising MVAS, methods of using crystals comprising
MvaS, a crystal structure of MvaS, and methods of using the crystal
structure.
BACKGROUND OF THE INVENTION
A general approach to designing inhibitors that are selective for a
given protein is to determine how a putative inhibitor interacts
with a three-dimensional structure of that protein. For this reason
it is useful to obtain the protein in crystalline form and perform
X-ray diffraction techniques to determine the protein's
three-dimensional structure coordinates. Various methods for
preparing crystalline proteins are known in the art.
Once protein crystals are produced, crystallographic data can be
generated using the crystals to provide useful structural
information that assists in the design of small molecules that bind
to the active site of the protein and inhibit the protein's
activity in vivo. If the protein is crystallized as a complex with
a ligand, one can determine both the shape of the protein's binding
pocket when bound to the ligand, as well as the amino acid residues
that are capable of close contact with the ligand. By knowing the
shape and amino acid residues comprised in the binding pocket, one
may design new ligands that will interact favorably with the
protein. With such structural information, available computational
methods may be used to predict how strong the ligand binding
interaction will be. Such methods aid in the design of inhibitors
that bind strongly, as well as selectively to the protein.
SUMMARY OF THE INVENTION
The present invention is directed to crystals comprising MvaS and
particularly crystals comprising MvaS that have sufficient size and
quality to obtain useful information about the structural
properties of MvaS and molecules or complexes that may associate
with MvaS.
In one embodiment, a composition is provided that comprises a
protein in crystalline form wherein at least a portion of the
protein has 55%, 65%, 75%, 85%, 90%, 95%, 97%, 99% or greater
identity with residues 2 384 of SEQ. ID No. 1.
In one variation, the protein has activity characteristic of MvaS.
For example, the protein may optionally be inhibited by inhibitors
of wild type MvaS. The protein crystal may also diffract X-rays for
a determination of structure coordinates to a resolution of 4
.ANG., 3.5 .ANG., 3.0 .ANG. or less.
In one variation, the protein crystal has a crystal lattice in a
P2.sub.12.sub.12.sub.1 space group. The protein crystal may also
have a crystal lattice having unit cell dimensions, +/-5%, of
a=68.7 b=79.6 c=150.2, .alpha.=.beta.=.gamma.=90.
The present invention is also directed to crystallizing MvaS. The
present invention is also directed to the conditions useful for
crystallizing MvaS. It should be recognized that a wide variety of
crystallization methods can be used in combination with the
crystallization conditions to form crystals comprising MvaS
including, but not limited to, vapor diffusion, batch, dialysis,
and other methods of contacting the protein solution for the
purpose of crystallization.
The present invention is also directed to crystallizing MvaS. The
present invention is also directed to the conditions useful for
crystallizing MvaS. It should be recognized that a wide variety of
crystallization methods can be used in combination with the
crystallization conditions to form crystals comprising MvaS
including, but not limited to, vapor diffusion, batch, dialysis,
and other methods of contacting the protein solution for the
purpose of crystallization.
In one embodiment, a method is provided for forming crystals of a
protein comprising: forming a crystallization volume comprising: a
protein wherein at least a portion of the protein has 55%, 65%,
75%, 85%, 90%, 95%, 97%, 99% or greater identity with residues 2
384 of SEQ. ID No. 1; and storing the crystallization volume under
conditions suitable for crystal formation.
In one embodiment, a method is provided for forming crystals of a
protein comprising: forming a crystallization volume comprising: a
protein that has at least 55% identity with residues 2 384 of SEQ.
ID No.1 in a concentration between 1 mg/ml and 100 mg/ml; 5 50% w/v
of precipitant wherein the precipitant comprises one or more
members of the group consisting of PEG or PEG MME having a
molecular weight between 200 and 10000; optionally 0.05 to 2.5M
additives wherein the additives comprise a monovalent and/or
divalent salt (for example, sodium, lithium, magnesium, calcium,
and the like); and storing the crystallization volume under
conditions suitable for crystal formation. The method also
optionally further includes performing the crystallization at a
temperature between 1.degree. C. 37.degree. C.
The method may optionally further comprise forming a protein
crystal that has a crystal lattice in a P2.sub.12.sub.12.sub.1,
space group. The method also optionally further comprises forming a
protein crystal that has a crystal lattice having unit cell
dimensions, +/-5%, a=68.7 b=79.6 c=150.2,
.alpha.=.beta.=.gamma.=90. The invention also relates to protein
crystals formed by these methods.
The present invention is also directed to a composition comprising
an isolated protein that comprises or consists of one or more of
the protein sequence(s) of MvaS taught herein for crystallizing
MvaS. The present invention is also directed to a composition
comprising an isolated nucleic acid molecule that comprises or
consists of the nucleotides for expressing the protein sequence of
MvaS taught herein for crystallizing MvaS.
The present invention is also directed to an expression vector that
may be used to express the isolated proteins taught herein for
crystallizing MvaS.
The present invention is also directed to an expression vector that
may be used to express the isolated proteins taught herein for
crystallizing MvaS. In one variation, the expression vector
comprises a promoter that promotes expression of the isolated
protein.
The present invention is also directed to a cell line transformed
or transfected by an isolated nucleic acid molecule or expression
vector of the present invention.
The present invention is also directed to structure coordinates for
MvaS as well as structure coordinates that are comparatively
similar to these structure coordinates. It is noted that these
comparatively similar structure coordinates may encompass proteins
with similar sequences and/or structures, such as ketoacyl-ACP
synthases. For example, machine-readable data storage media is
provided having data storage material encoded with machine-readable
data that comprises structure coordinates that are comparatively
similar to the structure coordinates of MvaS. The present invention
is also directed to a machine readable data storage medium having
data storage material encoded with machine readable data, which,
when read by an appropriate machine, can display a three
dimensional representation of all or a portion of a structure of
MvaS or a model that is comparatively similar to the structure of
all or a portion of MvaS.
Various embodiments of machine readable data storage medium are
provided that comprise data storage material encoded with machine
readable data. The machine readable data comprises: structure
coordinates that have a root mean square deviation equal to or less
than the RMSD value specified in Columns 3, 4 or 5 of Table 1 when
compared to the structure coordinates of FIG. 3, the root mean
square deviation being calculated such that the portion of amino
acid residues specified in Column 2 of Table 1 of each set of
structure coordinates are superimposed and the root mean square
deviation is based only on those amino acid residues in the
structure coordinates that are also present in the portion of the
protein specified in specified in Column 1 of Table 1. The amino
acids being overlayed and compared need not to be identical when
the RMSD calculation is performed on alpha carbons and main chain
atoms but the amino acids being overlayed and compared must have
identical side chains when the RMSD calculation is performed on all
non-hydrogen atoms.
For example, in one embodiment where the comparison is based on the
4 Angstrom set of amino acid residues (Column 1) and is based on
superimposing alpha-carbon atoms (Column 2), the structure
coordinates may have a root mean square deviation equal to or less
than 0.98 .ANG., 0.65 .ANG., or 0.49 .ANG. when compared to the
structure coordinates of FIG. 3.
TABLE-US-00001 TABLE 1 AA RESIDUES TO PORTION OF EACH AA USE TO
PERFORM RESIDUE USED TO RMSD VALUE RMSD PERFORM RMSD LESS THAN
COMPARISON COMPARISON OR EQUAL TO Table 2 alpha-carbon atoms.sup.1
0.98 0.65 0.49 (4 Angstrom set) main-chain atoms.sup.1 0.91 0.61
0.45 all non-hydrogen.sup.2 1.10 0.71 0.54 Table 3 alpha-carbon
atoms.sup.1 0.89 0.60 0.45 (7 Angstrom set) main-chain atoms.sup.1
0.84 0.56 0.42 all non-hydrogen.sup.2 0.99 0.66 0.49 Table 4
alpha-carbon atoms.sup.1 0.98 0.65 0.49 (10 Angstrom set)
main-chain atoms.sup.1 0.95 0.64 0.48 all non-hydrogen.sup.2 1.11
0.74 0.56 2-384 of alpha-carbon atoms.sup.1 1.30 0.86 0.65 SEQ. ID
No. 1 main-chain atoms.sup.1 1.31 0.87 0.65 all non-hydrogen.sup.2
1.42 0.94 0.71 .sup.1the RMSD computed between the atoms of all
amino acids that are common to both the target and the reference in
the aligned and superposed structure. The amino acids need not to
be identical. .sup.2the RMSD computed only between identical amino
acids, which are common to both the target and the reference in the
aligned and superposed structure.
The present invention is also directed to a three-dimensional
structure of all or a portion of MvaS. This three-dimensional
structure may be used to identify binding sites, to provide mutants
having desirable binding properties, and ultimately, to design,
characterize, or identify ligands capable of interacting with MvaS.
Ligands that interact with MvaS may be any type of atom, compound,
protein or chemical group that binds to or otherwise associates
with the protein. Examples of types of ligands include natural
substrates for MvaS, inhibitors of MvaS, and heavy atoms. The
inhibitors of MvaS may optionally be used as drugs to treat
therapeutic indications by modifying the in vivo activity of
MvaS.
In various embodiments, methods are provided for displaying a three
dimensional representation of a structure of a protein
comprising:
taking machine readable data comprising structure coordinates that
have a root mean square deviation equal to or less than the RMSD
value specified in Columns 3, 4 or 5 of Table 1 when compared to
the structure coordinates of FIG. 3, the root mean square deviation
being calculated such that the portion of amino acid residues
specified in Column 2 of Table 1 of each set of structure
coordinates are superimposed and the root mean square deviation is
based only on those amino acid residues in the structure
coordinates that are also present in the portion of the protein
specified in specified in Column 1 of Table 1;
computing a three dimensional representation of a structure based
on the structure coordinates; and
displaying the three dimensional representation.
The present invention is also directed to a method for solving a
three-dimensional crystal structure of a target protein using the
structure of MvaS.
In various embodiments, computational methods are provided
comprising: taking machine readable data comprising structure
coordinates that have a root mean square deviation equal to or less
than the RMSD value specified in Columns 3, 4 or 5 of Table 1 when
compared to the structure coordinates of FIG. 3, the root mean
square deviation being calculated such that the portion of amino
acid residues specified in Column 2 of Table 1 of each set of
structure coordinates are superimposed and the root mean square
deviation is based only on those amino acid residues in the
structure coordinates that are also present in the portion of the
protein specified in specified in Column 1 of Table 1;
computing phases based on the structural coordinates;
computing an electron density map based on the computed phases;
and
determining a three-dimensional crystal structure based on the
computed electron density map.
In various embodiments, computational methods are provided
comprising: taking an X-ray diffraction pattern of a crystal of the
target protein; and computing a three-dimensional electron density
map from the X-ray diffraction pattern by molecular replacement,
wherein structure coordinates used as a molecular replacement model
comprise structure coordinates that have a root mean square
deviation equal to or less than the RMSD value specified in Columns
3, 4 or 5 of Table 1 when compared to the structure coordinates of
FIG. 3, the root mean square deviation being calculated such that
the portion of amino acid residues specified in Column 2 of Table 1
of each set of structure coordinates are superimposed and the root
mean square deviation is based only on those amino acid residues in
the structure coordinates that are also present in the portion of
the protein specified in specified in Column 1 of Table 1.
These methods may optionally further comprise determining a
three-dimensional crystal structure based upon the computed
three-dimensional electron density map.
The present invention is also directed to using a crystal structure
of MvaS, in particular the structure coordinates of MvaS and the
surface contour defined by them, in methods for screening,
designing, or optimizing molecules or other chemical entities that
interact with and preferably inhibit MvaS.
One skilled in the art will appreciate the numerous uses of the
inventions described herein, particularly in the areas of drug
design, screening and optimization of drug candidates, as well as
in determining additional unknown crystal structures. For example,
a further aspect of the present invention relates to using a
three-dimensional crystal structure of all or a portion of MvaS
and/or its structure coordinates to evaluate the ability of
entities to associate with MvaS. The entities may be any entity
that may function as a ligand and thus may be any type of atom,
compound, protein (such as antibodies) or chemical group that can
bind to or otherwise associate with a protein.
In various embodiments, methods are provided for evaluating a
potential of an entity to associate with a protein comprising:
creating a computer model of a protein structure using structure
coordinates that comprise structure coordinates that have a root
mean square deviation equal to or less than the RMSD value
specified in Columns 3, 4 or 5 of Table 1 when compared to the
structure coordinates of FIG. 3, the root mean square deviation
being calculated such that the portion of amino acid residues
specified in Column 2 of Table 1 of each set of structure
coordinates are superimposed and the root mean square deviation is
based only on those amino acid residues in the structure
coordinates that are also present in the portion of the protein
specified in specified in Column 1 of Table 1;
performing a fitting operation between the entity and the computer
model; and
analyzing results of the fitting operation to quantify an
association between the entity and the model.
In other embodiments, methods are provided for identifying entities
that can associate with a protein comprising: generating a
three-dimensional structure of a protein using structure
coordinates that comprise structure coordinates that have a root
mean square deviation equal to or less than the RMSD value
specified in Columns 3, 4 or 5 of Table 1 when compared to the
structure coordinates of FIG. 3, the root mean square deviation
being calculated such that the portion of amino acid residues
specified in Column 2 of Table 1 of each set of structure
coordinates are superimposed and the root mean square deviation is
based only on those amino acid residues in the structure
coordinates that are also present in the portion of the protein
specified in specified in Column 1 of Table 1; and
employing the three-dimensional structure to design or select an
entity that can associate with the protein; and contacting the
entity with a protein wherein at least a portion of the protein has
55%, 65%, 75%, 85%, 90%, 95%, 97%, 99% or greater identity with
residues 2 384 of SEQ. ID No. 1.
In other embodiments, methods are provided for identifying entities
that can associate with a protein comprising:
generating a three-dimensional structure of a protein using
structure coordinates that comprise structure coordinates that have
a root mean square deviation equal to or less than the RMSD value
specified in Columns 3, 4 or 5 of Table 1 when compared to the
structure coordinates of FIG. 3, the root mean square deviation
being calculated such that the portion of amino acid residues
specified in Column 2 of Table 1 of each set of structure
coordinates are superimposed and the root mean square deviation is
based only on those amino acid residues in the structure
coordinates that are also present in the portion of the protein
specified in specified in Column 1 of Table 1; and
employing the three-dimensional structure to design or select an
entity that can associate with the protein.
In other embodiments, methods are provided for identifying entities
that can associate with a protein comprising:
computing a computer model for a protein binding pocket, at least a
portion of the computer model having a surface contour that has a
root mean square deviation equal to or less than a given RMSD value
specified in Columns 3, 4 or 5 of Table 1 when the coordinates used
to compute the surface contour are compared to the structure
coordinates of FIG. 3, wherein (a) the root mean square deviation
is calculated by the calculation method set forth herein, (b) the
portion of amino acid residues associated with the given RMSD value
in Table 1 (specified in Column 2 of Table 1) are superimposed
according to the RMSD calculation, and (c) the root mean square
deviation is calculated based only on those amino acid residues
present in both the protein being modeled and the portion of the
protein associated with the given RMSD in Table 1 (specified in
Column 1 of Table 1);
employing the computer model to design or select an entity that can
associate with the protein; and contacting the entity with a
protein wherein at least a portion of the protein has 55%, 65%,
75%, 85%, 90%, 95%, 97%, 99% or greater identity with residues 2
384 of SEQ. ID No. 1.
In other embodiments, methods are provided for identifying entities
that can associate with a protein comprising:
computing a computer model for a protein binding pocket, at least a
portion of the computer model having a surface contour that has a
root mean square deviation equal to or less than a given RMSD value
specified in Columns 3, 4 or 5 of Table 1 when the coordinates used
to compute the surface contour are compared to the structure
coordinates of FIG. 3, wherein (a) the root mean square deviation
is calculated by the calculation method set forth herein, (b) the
portion of amino acid residues associated with the given RMSD value
in Table 1 (specified in Column 2 of Table 1) are superimposed
according to the RMSD calculation, and (c) the root mean square
deviation is calculated based only on those amino acid residues
present in both the protein being modeled and the portion of the
protein associated with the given RMSD in Table 1 (specified in
Column 1 of Table 1); and
employing the computer model to design or select an entity that can
associate with the protein.
In other embodiments, methods are provided for evaluating the
ability of an entity to associate with a protein, the method
comprising:
constructing a computer model defined by structure coordinates that
have a root mean square deviation equal to or less than the RMSD
value specified in Columns 3, 4 or 5 of Table 1 when compared to
the structure coordinates of FIG. 3, the root mean square deviation
being calculated such that the portion of amino acid residues
specified in Column 2 of Table 1 of each set of structure
coordinates are superimposed and the root mean square deviation is
based only on those amino acid residues in the structure
coordinates that are also present in the portion of the protein
specified in specified in Column 1 of Table 1; and
selecting an entity to be evaluated by a method selected from the
group consisting of (i) assembling molecular fragments into the
entity, (ii) selecting an entity from a small molecule database,
(iii) de novo ligand design of the entity, and (iv) modifying a
known ligand for MvaS, or a portion thereof; performing a fitting
program operation between computer models of the entity to be
evaluated and the binding pocket in order to provide an
energy-minimized configuration of the entity in the binding pocket;
and evaluating the results of the fitting operation to quantify the
association between the entity and the binding pocket model in
order to evaluate the ability of the entity to associate with the
binding pocket.
In other embodiments, methods are provided for evaluating the
ability of an entity to associate with a protein, the method
comprising:
computing a computer model for a protein binding pocket, at least a
portion of the computer model having a surface contour that has a
root mean square deviation equal to or less than a given RMSD value
specified in Columns 3, 4 or 5 of Table 1 when the coordinates used
to compute the surface contour are compared to the structure
coordinates of FIG. 3, wherein (a) the root mean square deviation
is calculated by the calculation method set forth herein, (b) the
portion of amino acid residues associated with the given RMSD value
in Table 1 (specified in Column 2 of Table 1) are superimposed
according to the RMSD calculation, and (c) the root mean square
deviation is calculated based only on those amino acid residues
present in both the protein being modeled and the portion of the
protein associated with the given RMSD in Table 1 (specified in
Column 1 of Table 1); and
selecting an entity to be evaluated by a method selected from the
group consisting of (i) assembling molecular fragments into the
entity, (ii) selecting an entity from a small molecule database,
(iii) de novo ligand design of the entity, and (iv) modifying a
known ligand for MvaS, or a portion thereof; performing a fitting
program operation between computer models of the entity to be
evaluated and the binding pocket in order to provide an
energy-minimized configuration of the entity in the binding pocket;
and evaluating the results of the fitting operation to quantify the
association between the entity and the binding pocket model in
order to evaluate the ability of the entity to associate with the
binding pocket.
In regard to each of these embodiments, the protein may optionally
have activity characteristic of MvaS. For example, the protein may
optionally be inhibited by inhibitors of wild type MvaS.
In another embodiment, a method is provided for identifying an
entity that associates with a protein comprising: taking structure
coordinates from diffraction data obtained from a crystal of a
protein wherein at least a portion of the protein has 55%, 65%,
75%, 85%, 90%, 95%, 97%, 99% or greater identity with residues 2
384 of SEQ. ID No. 1; and performing rational drug design using a
three dimensional structure that is based on the obtained structure
coordinates.
The protein crystals may optionally have a crystal lattice with a
P2.sub.12.sub.12.sub.1, space group and unit cell dimensions,
+/-5%, of a=68.7 b=79.6 c=150.2, .alpha.=.beta.=.gamma.=90.
The method may optionally further comprise selecting one or more
entities based on the rational drug design and contacting the
selected entities with the protein. The method may also optionally
further comprise measuring an activity of the protein when
contacted with the one or more entities. The method also may
optionally further comprise comparing activity of the protein in a
presence of and in the absence of the one or more entities; and
selecting entities where activity of the protein changes depending
whether a particular entity is present. The method also may
optionally further comprise contacting cells expressing the protein
with the one or more entities and detecting a change in a phenotype
of the cells when a particular entity is present.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates SEQ. ID Nos. 1 and 2 referred to in this
application.
FIG. 2 illustrates crystal of MvaS corresponding to SEQ. ID No. 1,
having a crystal lattice in a P2.sub.12.sub.12.sub.1, space group
and unit cell dimensions, +/-5%, of a=68.7 b=79.6 c=150.2,
.alpha.=.beta.=.gamma.=90.
FIG. 3 lists a set of atomic structure coordinates for MvaS as
derived by X-ray crystallography from a crystal that comprises the
protein. The following abbreviations are used in FIG. 3: "X, Y, Z"
crystallographically define the atomic position of the element
measured; "B" is a thermal factor that measures movement of the
atom around its atomic center; "Occ" is an occupancy factor that
refers to the fraction of the molecules in which each atom occupies
the position specified by the coordinates (a value of "1" indicates
that each atom has the same conformation, i.e., the same position,
in all molecules of the crystal).
FIG. 4 illustrates a ribbon diagram overview of the structure of
MvaS, highlighting secondary structural elements of the
protein.
FIG. 5 illustrates the MvaS binding site of MvaS based on the
determined crystal structure for the molecule in the asymmetric
unit corresponding to the coordinates shown in FIG. 3.
FIG. 6 illustrates a system that may be used to carry out
instructions for displaying a crystal structure of MvaS encoded on
a storage medium.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to 3-Hydroxy-3-methylglutaryl-CoA
(HMG-CoA) and more specifically to MvaS also known as HMG-CoA
Synthase. Provided is MvaS in crystalline form, methods of forming
crystals comprising MvaS, methods of using crystals comprising
MvaS, a crystal structure of MvaS, and methods of using the crystal
structure.
In describing protein structure and function herein, reference is
made to amino acids comprising the protein. The amino acids may
also be referred to by their conventional abbreviations;
A=Ala=Alanine; T=Thr=Threonine; V=Val=Valine; C=Cys=Cysteine;
L=Leu=Leucine; Y=Tyr=Tyrosine; I=Ile=Isoleucine; N=Asn=Asparagine;
P=Pro=Proline; Q=Gln=Glutamine; F=Phe=Phenylalanine; D=Asp=Aspartic
Acid; W=Trp=Tryptophan; E=Glu=Glutamic Acid;
M=MSE=Selenomethionine; K=Lys=Lysine; G=Gly=Glycine;
R=Arg=Arginine; S=Ser=Serine; and H=His=Histidine.
1. MvaS
The protein MvaS is an acyl-keto synthase that condenses acetyl-CoA
and acetoacetyl-CoA to from HMG-CoA. In humans, the enzyme
catalyzes a committed step in the pathways for isoprenoid,
cholesterol, and ketone body production. The importance of the
enzyme is underscored by the observed regulation of various
isozymes (Quaint P. A. (1994) Essays Biochem, 28, 13 25) as well
the role the enzyme in fasting hypoketotic coma (N Engl J Med. 1997
Oct. 23;337(17):1203 7)
MvaS is also found in gram-positive pathogenic bacteria such and
Enterococcus facaelis. As such knowledge of the 3-dimensional
structure of a bacterial MvaS enzyme will facilitate the
development of compounds that inhibit the growth of pathogens
dependent on the mevalonate pathway for the synthesis of
isoprenoids.
In one embodiment, MvaS comprises the wild-type form of full length
MvaS, set forth herein as SEQ. ID No. 1 (GenBank Accession Number
AF290092).
In another embodiment, MvaS comprises residues 2 384 of SEQ. ID No.
1 which comprises the active site domain of wild-type MvaS that is
represented in the set of structural coordinates shown in FIG.
3.
It should be recognized that the invention may be readily extended
to various variants of wild-type MvaS and variants of fragments
thereof. In another embodiment, MvaS comprises a sequence wherein
at least a portion of the sequence has 55%, 65%, 75%, 85%, 90%,
95%, 97%, 99% or greater identity with SEQ. ID No. 1.
It is also noted that the above sequences of MvaS are also intended
to encompass isoforms, mutants and fusion proteins of these
sequences. An example of a fusion protein is provided by SEQ. ID
No. 1, which includes a 6 residue C-terminal tag (6 residues are
histidine) that may be used to facilitate purification of the
protein.
With the crystal structure provided herein, it is now known where
amino acid residues are positioned in the structure. As a result,
the impact of different substitutions can be more easily predicted
and understood.
For example, based on the crystal structure, applicants have
determined that the MvaS amino acids shown in Table 2 encompass a
4-Angstrom radius around the MvaS active site and thus likely to
interact with any active site inhibitor of MvaS. Applicants have
also determined that the amino acids of Table 3 encompass a
7-Angstrom radius around the MvaS active site. Further it has been
determined that the amino acids of Table 4 encompass a 10-Angstrom
radius around the MvaS active site. It is noted that there is one
MvaS molecule in the asymmetric unit, referred to as chain A.
Structural coordinates appear in FIG. 3. It is noted that the
sequence and structure of the residues in the active site may also
be conserved and hence pertinent to other MvaS and homologs.
One or more of the sets of amino acids set forth in the tables is
preferably conserved in a variant of MvaS. Hence, MvaS may
optionally comprise a sequence wherein at least a portion of the
sequence has 55%, 65%, 75%, 85%, 90%, 95%, 97%, 99% or greater
identity with any one of the above sequences (e.g., all of SEQ. ID
No. 1 or residues 2 384 of SEQ. ID No. 1) where at least the
residues shown in Tables 2, 3, and/or 4 are conserved with the
exception of 0, 1, 2, 3, or 4 residues. It should be recognized
that one might optionally vary some of the binding site residues in
order to determine the effect such changes have on structure or
activity.
TABLE-US-00002 TABLE 2 Amino Acids encompassed by a 4-Angstrom
radius around the MvaS active site. ASP 29 GLY 31 LYS 32 GLY 36 GLU
79 ALA 110 CYS 111 TYR 143 GLY 148 GLY 149 THR 152 PHE 185 VAL 196
GLY 198 SER 201 ASN 202 TYR 205 HIS 233 PRO 235 TYR 236 MSE 239 LYS
242 ASN 275 TYR 306 GLY 307 SER 308
TABLE-US-00003 TABLE 3 Amino Acids encompassed by a 7-Angstrom
radius around the MvaS active site. VAL 28 ASP 29 PRO 30 GLY 31 LYS
32 PHE 33 HIS 34 ILE 35 GLY 36 ILE 37 GLU 79 GLU 109 ALA 110 CYS
111 TYR 112 ASP 139 TYR 143 SER 147 GLY 148 GLY 149 GLU 150 PRO 151
THR 152 ASP 184 PHE 185 TRP 186 PRO 194 VAL 196 ASP 197 GLY 198 PRO
199 LEU 200 SER 201 ASN 202 GLU 203 THR 204 TYR 205 ILE 206 HIS 233
ILE 234 PRO 235 TYR 236 LYS 238 MSE 239 LYS 242 ASN 275 TYR 277 THR
278 SER 280 TYR 306 GLY 307 SER 308 GLY 309
TABLE-US-00004 TABLE 4 Amino Acids encompassed by a 10-Angstrom
radius around the MvaS active site. MSE 19 ALA 23 ASN 27 VAL 28 ASP
29 PRO 30 GLY 31 LYS 32 PHE 33 HIS 34 ILE 35 GLY 36 ILE 37 GLY 38
GLN 39 MSE 42 THR 78 GLU 79 SER 80 LYS 108 GLU 109 ALA 110 CYS 111
TYR 112 GLY 113 ALA 114 ASP 139 ILE 140 ALA 141 LYS 142 TYR 143 GLY
144 ASN 146 SER 147 GLY 148 GLY 149 GLU 150 PRO 151 THR 152 GLN 153
GLY 154 ILE 182 TYR 183 ASP 184 PHE 185 TRP 186 ARG 187 PRO 194 MSE
195 VAL 196 ASP 197 GLY 198 PRO 199 LEU 200 SER 201 ASN 202 GLU 203
THR 204 TYR 205 ILE 206 GLN 207 SER 208 PHE 232 HIS 233 ILE 234 PRO
235 TYR 236 THR 237 LYS 238 MSE 239 GLY 240 LYS 241 LYS 242 ALA 243
LEU 245 TYR 263 GLY 274 ASN 275 LEU 276 TYR 277 THR 278 GLY 279 SER
280 LEU 281 PHE 304 SER 305 TYR 306 GLY 307 SER 308 GLY 309 ALA 310
VAL 311 ALA 312
With the benefit of the crystal structure and guidance provided by
Tables 2, 3 and 4, a wide variety of MvaS variants (e.g.,
insertions, deletions, substitutions, etc.) that fall within the
above specified identity ranges may be designed and manufactured
utilizing recombinant DNA techniques well known to those skilled in
the art, particularly in view of the knowledge of the crystal
structure provided herein. These modifications can be used in a
number of combinations to produce the variants. The present
invention is useful for crystallizing and then solving the
structure of the range of variants of MvaS.
Variants of MvaS may be insertional variants in which one or more
amino acid residues are introduced into a predetermined site in the
MvaS sequence. For instance, insertional variants can be fusions of
heterologous proteins or polypeptides to the amino or carboxyl
terminus of the subunits.
Variants of MvaS also may be substitutional variants in which at
least one residue has been removed and a different residue inserted
in its place. Non-natural amino acids (i.e. amino acids not
normally found in native proteins), as well as isosteric analogs
(amino acid or otherwise) may optionally be employed in
substitutional variants. Examples of suitable substitutions are
well known in the art, such as the Glu.fwdarw.Asp, Asp.fwdarw.Glu,
Ser.fwdarw.Cys, and Cys.fwdarw.Ser for example.
Another class of variants is deletional variants, which are
characterized by the removal of one or more amino acid residues
from the MvaS sequence.
Other variants may be produced by chemically modifying amino acids
of the native protein (e.g., diethylpyrocarbonate treatment that
modifies histidine residues). Preferred are chemical modifications
that are specific for certain amino acid side chains. Specificity
may also be achieved by blocking other side chains with antibodies
directed to the side chains to be protected. Chemical modification
includes such reactions as oxidation, reduction, amidation,
deamidation, or substitution of bulky groups such as
polysaccharides or polyethylene glycol.
Exemplary modifications include the modification of lysinyl and
amino terminal residues by reaction with succinic or other
carboxylic acid anhydrides. Modification with these agents has the
effect of reversing the charge of the lysinyl residues. Other
suitable reagents for modifying amino-containing residues include
imidoesters such as methyl picolinimidate; pyridoxal phosphate;
pyridoxal chloroborohydride; trinitrobenzenesulfonic acid;
O-methylisourea, 2,4-pentanedione; and transaminaseN catalyzed
reaction with glyoxylate, and N-hydroxysuccinamide esters of
polyethylene glycol or other bulky substitutions.
Arginyl residues may be modified by reaction with a number of
reagents, including phenylglyoxal, 2,3-butanedione,
1,2-cyclohexanedione, and ninhydrin. Modification of arginine
residues requires that the reaction be performed in alkaline
conditions because of the high pK.sub.a, of the guanidine
functional group. Furthermore, these reagents may react with the
groups of lysine as well as the arginine epsilon-amino group.
Tyrosyl residues may also be modified to introduce spectral labels
into tyrosyl residues by reaction with aromatic diazonium compounds
or tetranitromethane, forming O-acetyl tyrosyl species and 3-nitro
derivatives, respectively. Tyrosyl residues may also be iodinated
using .sup.125I or .sup.131I to prepare labeled proteins for use in
radioimmunoassays.
Carboxyl side groups (aspartyl or glutamyl) may be selectively
modified by reaction with carbodiimides or they may be converted to
asparaginyl and glutaminyl residues by reaction with ammonium ions.
Conversely, asparaginyl and glutaminyl residues may be deamidated
to the corresponding aspartyl or glutamyl residues, respectively,
under mildly acidic conditions. Either form of these residues falls
within the scope of this invention.
Other modifications that may be formed include the hydroxylation of
proline and lysine, phosphorylation of hydroxyl groups of seryl or
threonyl groups of lysine, arginine and histidine side chains (T.
E. Creighton, Proteins: Structure and Molecular Properties, W.H.
Freeman & Co., San Francisco, pp. 79 86, 1983), acetylation of
the N-terminal amine and amidation of any C-terminal carboxyl
group.
As can be seen, modifications of the nucleic sequence encoding MvaS
may be accomplished by a variety of well-known techniques, such as
site-directed mutagenesis (see, Gillman and Smith, Gene 8:81 97
(1979) and Roberts, S. et al., Nature 328:731 734 (1987)). When
modifications are made, these modifications may optionally be
evaluated for there affect on a variety of different properties
including, for example, solubility, crystallizability and a
modification to the protein's structure and activity.
In one variation, the variant and/or fragment of wild-type MvaS is
functional in the sense that the resulting protein is capable of
associating with at least one same chemical entity that is also
capable of selectively associating with a protein comprising the
wild-type MvaS (e.g., residues 2 384 of SEQ. ID No. 1) since this
common associative ability evidences that at least a portion of the
native structure has been conserved.
It is noted the activity of the native protein need not necessarily
be conserved. Rather, amino acid substitutions, additions or
deletions that interfere with native activity but which do not
significantly alter the three-dimensional structure of the domain
are specifically contemplated by the invention. Crystals comprising
such variants of MvaS, and the atomic structure coordinates
obtained there from, can be used to identify compounds that bind to
the native domain. These compounds may affect the activity of the
native domain.
Amino acid substitutions, deletions and additions that do not
significantly interfere with the three-dimensional structure of
MvaS will depend, in part, on the region where the substitution,
addition or deletion occurs in the crystal structure. These
modifications to the protein can now be made far more intelligently
with the crystal structure information provided herein. In highly
variable regions of the molecule, non-conservative substitutions as
well as conservative substitutions may be tolerated without
significantly disrupting the three-dimensional structure of the
molecule. In highly conserved regions, or regions containing
significant secondary structure, conservative amino acid
substitutions are preferred.
Conservative amino acid substitutions are well known in the art,
and include substitutions made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity and/or
the amphipathic nature of the amino acid residues involved. For
example, negatively charged amino acids include aspartic acid and
glutamic acid; positively charged amino acids include lysine and
arginine; amino acids with uncharged polar head groups having
similar hydrophilicity values include the following: leucine,
isoleucine, valine; glycine, alanine; asparagine, glutamine;
serine, threonine; phenylalanine, tyrosine. Other conservative
amino acid substitutions are well known in the art.
It should be understood that the protein may be produced in whole
or in part by chemical synthesis. As a result, the selection of
amino acids available for substitution or addition is not limited
to the genetically encoded amino acids. Indeed, mutants may
optionally contain non-genetically encoded amino acids.
Conservative amino acid substitutions for many of the commonly
known non-genetically encoded amino acids are well known in the
art. Conservative substitutions for other amino acids can be
determined based on their physical properties as compared to the
properties of the genetically encoded amino acids.
In some instances, it may be particularly advantageous or
convenient to substitute, delete and/or add amino acid residues in
order to provide convenient cloning sites in cDNA encoding the
polypeptide, to aid in purification of the polypeptide, etc. Such
substitutions, deletions and/or additions which do not
substantially alter the three dimensional structure of MvaS will be
apparent to those having skills in the art, particularly in view of
the three dimensional structure of MvaS provided herein.
2. Cloning, Expression and Purification of MvaS
The gene encoding MvaS can be isolated from RNA, cDNA or cDNA
libraries. In this case, the portion of the gene encoding amino
acid residues 2 384 (SEQ. ID No. 1) was isolated and is shown as
SEQ. ID No. 2. It is noted that the gene was modified to include a
C-terminal 6 residue polyhistidine tag.
Construction of expression vectors and recombinant proteins from
the DNA sequence encoding MvaS may be performed by various methods
well known in the art. For example, these techniques may be
performed according to Sambrook et al., Molecular Cloning--A
Laboratory Manual, Cold Spring Harbor, N.Y. (1989), and Kriegler,
M., Gene Transfer and Expression, A Laboratory Manual, Stockton
Press, New York (1990).
A variety of expression systems and hosts may be used for the
expression of MvaS. Example 1 provides one such expression
system.
Once expressed, purification steps are employed to produce MvaS in
a relatively homogeneous state. In general, a higher purity
solution of a protein increases the likelihood that the protein
will crystallize. Typical purification methods include the use of
centrifugation, partial fractionation, using salt or organic
compounds, dialysis, conventional column chromatography, (such as
ion exchange, molecular sizing chromatography, etc.), high
performance liquid chromatography (HPLC), and gel electrophoresis
methods (see, e.g., Deutcher, "Guide to Protein Purification" in
Methods in Enzymology (1990), Academic Press, Berkeley,
Calif.).
MvaS may optionally be affinity labeled during cloning, preferably
with a N-terminal six-histidine tag and rTev cleavage site, in
order to facilitate purification. With the use of an affinity
label, it is possible to perform a one-step purification process on
a purification column that has a unique affinity for the label. The
affinity label may be optionally removed after purification. These
and other purification methods are known and will be apparent to
one of skill in the art.
3. Crystallization & Crystals Comprising MvaS
One aspect of the present invention relates to methods for forming
crystals comprising MvaS as well as crystals comprising MvaS.
In one embodiment, a method for forming crystals comprising MvaS is
provided comprising forming a crystallization volume comprising
MvaS, one or more precipitants, optionally a buffer, optionally a
monovalent and/or divalent salt and optionally an organic solvent;
and storing the crystallization volume under conditions suitable
for crystal formation.
In yet another embodiment, a method for forming crystals comprising
MvaS is provided comprising forming a crystallization volume
comprising MvaS in solution comprising the components shown in
Table 5; and storing the crystallization volume under conditions
suitable for crystal formation.
TABLE-US-00005 TABLE 5 Precipitant 5 50% w/v of precipitant wherein
the precipitant comprises one or more members of the group
consisting of PEG MME having a molecular weight range between 1000
10000, PEG having a molecular weight range between 100 10000. 0.3
2.0 M Sodium, potassium or ammonium phosphate. pH pH 4 10. Buffers
that may be used include, but are not limited to tris, bicine,
phosphate, cacodylate, acetate, citrate, HEPES, PIPES, MES and
combinations thereof. Additives Optionally 0.05 to 2.5 M additives
wherein the additives comprise a monovalent and/or divalent salt
(for example, sodium, lithium, magnesium, calcium, and the like)
Protein Concentration 1 mg/ml 50 mg/ml Temperature 1.degree. C.
25.degree. C.
In yet another embodiment, a method for forming crystals comprising
MvaS is provided comprising forming a crystallization volume
comprising MvaS; introducing crystals comprising MvaS as nucleation
sites, and storing the crystallization volume under conditions
suitable for crystal formation.
Crystallization experiments may optionally be performed in volumes
commonly used in the art, for example typically 15, 10, 5, 2
microliters or less. It is noted that the crystallization volume
optionally has a volume of less than 1 microliter, optionally 500,
250, 150, 100, 50 or less nanoliters.
It is also noted that crystallization may be performed by any
crystallization method including, but not limited to batch,
dialysis and vapor diffusion (e.g., sitting drop and hanging drop)
methods. Micro, macro and/or streak seeding of crystals may also be
performed to facilitate crystallization.
It should be understood that forming crystals comprising MvaS and
crystals comprising MvaS according to the invention are not
intended to be limited to the wild type, full length MvaS shown in
SEQ. ID No. 1, fragments comprising residues 2 384 of SEQ. ID No. 1
and fragments comprising residues 2 384 of SEQ. ID No. 1. Rather,
it should be recognized that the invention may be extended to
various other fragments and variants of wild-type MvaS as described
above.
It should also be understood that forming crystals comprising MvaS
and crystals comprising MvaS according to the invention may be such
that MvaS is optionally complexed with one or more ligands and one
or more copies of the same ligand. The ligand used to form the
complex may be any ligand capable of binding to MvaS. In one
variation, the ligand is a natural substrate. In another variation,
the ligand is an inhibitor.
In one particular embodiment, MvaS crystals have a crystal lattice
in the P2.sub.12.sub.12.sub.1, space group. MvaS crystals may also
optionally have unit cell dimensions, +/-5%, of a=68.7 b=79.6
c=150.2, .alpha.=.beta.=.gamma.=90. MvaS crystals also preferably
are capable of diffracting X-rays for determination of atomic
coordinates to a resolution of 4 .ANG., 3.5 .ANG., 3.0 .ANG. or
better.
Crystals comprising MvaS may be formed by a variety of different
methods known in the art. For example, crystallizations may be
performed by batch, dialysis, and vapor diffusion (sitting drop and
hanging drop) methods. A detailed description of basic protein
crystallization setups may be found in McRee, D., Practical Protein
Crystallography, 2.sup.nd Ed. (1999), Academic Press Inc. Further
descriptions regarding performing crystallization experiments are
provided in Stevens, et al. (2000) Curr. Opin. Struct. Biol.:
10(5):558 63, and U.S. Pat. Nos. 6,296,673, 5,419,278, and
5,096,676.
In one variation, crystals comprising MvaS are formed by mixing
substantially pure MvaS with an aqueous buffer containing a
precipitant at a concentration just below a concentration necessary
to precipitate the protein. One suitable precipitant for
crystallizing MvaS is polyethylene glycol (PEG), which combines
some of the characteristics of the salts and other organic
precipitants (see, for example, Ward et al., J. Mol. Biol. 98:161,
1975, and McPherson, J. Biol. Chem. 251:6300, 1976).
During a crystallization experiment, water is removed by diffusion
or evaporation to increase the concentration of the precipitant,
thus creating precipitating conditions for the protein. In one
particular variation, crystals are grown by vapor diffusion in
hanging drops or sitting drops. According to these methods, a
protein/precipitant solution is formed and then allowed to
equilibrate in a closed container with a larger aqueous reservoir
having a precipitant concentration for producing crystals. The
protein/precipitant solution continues to equilibrate until
crystals grow.
By performing submicroliter volume sized crystallization
experiments, as detailed in U.S. Pat. No. 6,296,673, effective
crystallization conditions for forming crystals of a MvaS complex
were obtained. In order to accomplish this, systematic broad screen
crystallization trials were performed on an MvaS complex using the
sitting drop technique. In each experiment, a 100 nL mixture of
MvaS complex and precipitant was placed on a platform positioned
over a well containing 100 .mu.L of the precipitating solution.
Precipitate and crystal formation was detected in the sitting
drops. Fine screening was then carried out for those
crystallization conditions that appeared to produce precipitate
and/or crystal in the drops.
Based on the crystallization experiments that were performed, a
thorough understanding of how different crystallization conditions
affect MvaS crystallization was obtained. Based on this
understanding, a series of crystallization conditions were
identified that may be used to form crystals comprising MvaS. These
conditions are summarized in Table 5. A particular example of
crystallization conditions that may be used to form diffraction
quality crystals of the MvaS complex is detailed in Example 2. FIG.
2 illustrates crystals of the MvaS complex formed using the
crystallization conditions provided in Table 5.
One skilled in the art will recognize that the crystallization
conditions provided in Table 5 and Example 2 can be varied and
still yield protein crystals comprising MvaS. For example, it is
noted that variations on the crystallization conditions described
herein can be readily determined by taking the conditions provided
in Table 5 and performing fine screens around those conditions by
varying the type and concentration of the components in order to
determine additional suitable conditions for crystallizing MvaS,
variants of MvaS, and ligand complexes thereof.
Crystals comprising MvaS have a wide range of uses. For example,
now that crystals comprising MvaS have been produced, it is noted
that crystallizations may be performed using such crystals as a
nucleation site within a concentrated protein solution. According
to this variation, a concentrated protein solution is prepared and
crystalline material (microcrystals) is used to `seed` the protein
solution to assist nucleation for crystal growth. If the
concentrations of the protein and any precipitants are optimal for
crystal growth, the seed crystal will provide a nucleation site
around which a larger crystal forms. Given the ability to form
crystals comprising MvaS according to the present invention, the
crystals so formed can be used by this crystallization technique to
initiate crystal growth of other MvaS comprising crystals,
including MvaS complexed to other ligands.
As will be described herein in greater detail, crystals may also be
used to perform X-ray or neutron diffraction analysis in order to
determine the three-dimensional structure of MvaS and, in
particular, to assist in the identification of its active site.
Knowledge of the binding site region allows rational design and
construction of ligands including inhibitors. Crystallization and
structural determination of MvaS mutants having altered bioactivity
allows the evaluation of whether such changes are caused by general
structure deformation or by side chain alterations at the
substitution site.
4. X-Ray Data Collection and Structure Determination
Crystals comprising MvaS may be obtained as described above in
Section 3. As described herein, these crystals may then be used to
perform X-ray data collection and for structure determination.
In one embodiment, described in Example 2, crystals of MvaS were
obtained where MvaS has the sequence of residues shown in SEQ. ID
No. 1. These particular crystals were used to determine the three
dimensional structure of MvaS. However, it is noted that other
crystals comprising MvaS including different MvaS variants,
fragments, and complexes thereof may also be used.
Diffraction data was collected from cryocooled crystals (100K) of
MvaS at the Advanced Light Source (ALS) beam line 5.0.3 using an
ADSC Quantum CCD detector. The diffraction pattern of the MvaS
crystals displayed symmetry consistent with space group
P2.sub.12.sub.12.sub.1, with unit cell dimensions of a=68.7 b=79.6
c=150.2, .alpha.=.beta.=.gamma.=90 (+/-5%). Data were collected and
integrated to 2.0 .ANG. with the HKL2000 program package
(Otwinowski, Z. and Minor, W., Meth. Enzymol. 276:307 (1997).
The structure solution for MvaS in the space group
P2.sub.12.sub.12.sub.1, with unit cell dimensions of a=68.7 b=79.6
c=150.2, .alpha.=.beta.=.gamma.=90 (+/-5%) was obtained by rigid
body refinement method using the program REFMAC (Collaborative
Computational Project, N. The CCP4 Suite: Programs for Protein
Crystallography. Acta Crystallogr. D50, 760 763 (1994)), with the
coordinates of unliganded MvaS (unpublished results, (2002) Syrrx
Inc.) used as a search model. All subsequent crystallographic
calculations were performed using the CCP4 program package
(Collaborative Computational Project, N. The CCP4 Suite: Programs
for Protein Crystallography. Acta Crystallogr. D50, 760 763
(1994)). The rigid body solution was subjected to restrained
least-squares refinement using the maximum likelihood method as
implemented in REFMAC (Murshudov, G. N., Vagin, A. A. and Dodson E.
J. Acta Crystallogr D53:240 (1997)). Multiple rounds of manual
fitting of the MvaS sequence were performed with Xfit (McRee, D.
E., J. Struct. Biol. 125:156 (1999)). Manual fitting was
interspersed with restrained least-squares refinement in REFMAC
against data from 10.0 to 2.0 .ANG.. All stages of refinement were
carried with bulk solvent corrections and anisotropic scaling, and
excluded 5% of R.sub.free reflections for cross-validation. The
data collection and data refinement statistics are given in Table
6.
TABLE-US-00006 TABLE 6 Crystal data Space group
P2.sub.12.sub.12.sub.1 Unit cell dimensions a = 68.70 .ANG. b =
79.60 .ANG. c = 150.20 .ANG. Data collection X-ray source ALS BL
5.0.3 Wavelength [.ANG.] 1.00 Resolution [.ANG.] 2.00 Observations
(unique) 52889 Redundancy 4.7 Completeness overall (outer shell)
93.6 (59)% I/.sigma.(I) overall (outer shell) 24.4 (6.0)
R.sub.symm.sup.1 overall (outer shell) 0.070 (.116) Refinement
Reflections used 50122 R-factor 15.7% R.sub.free 19.6% r.m.s bonds
0.007 .ANG. r.m.s angles 1.33.degree.
During structure determination, where the unit cell dimensions were
a=68.7 b=79.6 c=150.2, .alpha.=.beta.=.gamma.=90, it was realized
that the asymmetric unit comprised two MvaS molecules. Structure
coordinates were determined for this complex and the resultant set
of structural coordinates from the refinement are presented in FIG.
3.
It is noted that the sequence of the structure coordinates
presented in FIG. 3 differ in some regards from the sequence shown
in SEQ. ID No. 1. Structure coordinates are not reported for
residues 1 and 384 391 in molecule A and residues 1 and 388 391 in
molecule B because the electron density obtained was insufficient
to identify their position. Those of skill in the art understand
that a set of structure coordinates (such as those in FIG. 3) for a
protein or a protein-complex or a portion thereof, is a relative
set of points that define a shape in three dimensions. Thus, it is
possible that an entirely different set of structure coordinates
could define a similar or identical shape. Moreover, slight
variations in the individual coordinates may have little effect on
overall shape. In terms of binding pockets, these variations would
not be expected to significantly alter the nature of ligands that
could associate with those pockets. The term "binding pocket" as
used herein refers to a region of the protein that, as a result of
its shape, favorably associates with a ligand.
These variations in coordinates may be generated because of
mathematical manipulations of the MvaS structure coordinates. For
example, the sets of structure coordinates shown in FIG. 3 could be
manipulated by crystallographic permutations of the structure
coordinates, fractionalization of the structure coordinates,
application of a rotation matrix, integer additions or subtractions
to sets of the structure coordinates, inversion of the structure
coordinates or any combination of the above.
Alternatively, modifications in the crystal structure due to
mutations, additions, substitutions, and/or deletions of amino
acids or other changes in any of the components that make up the
crystal could also account for variations in structure coordinates.
If such variations are within an acceptable standard error as
compared to the original coordinates, the resulting
three-dimensional shape should be considered to be the same. Thus,
for example, a ligand that bound to the active site binding pocket
of MvaS would also be expected to bind to another binding pocket
whose structure coordinates defined a shape that fell within the
acceptable error.
Various computational methods may be used to determine whether a
particular protein or a portion thereof (referred to here as the
"target protein"), typically the binding pocket, has a high degree
of three-dimensional spatial similarity to another protein
(referred to here as the "reference protein") against which the
target protein is being compared.
The process of comparing a target protein structure to a reference
protein structure may generally be divided into three steps: 1)
defining the equivalent residues and/or atoms for the target and
reference proteins, 2) performing a fitting operation between the
proteins; and 3) analyzing the results. These steps are described
in more detail below. All structure comparisons reported herein and
the structure comparisons claimed are intended to be based on the
particular comparison procedure described below.
Equivalent residues or atoms can be determined based upon an
alignment of primary sequences of the proteins, an alignment of
their structural domains or as a combination of both. Sequence
alignments generally implement the dynamic programming algorithm of
Needleman and Wunsch [J. Mol. Biol. 48: 442 453, 1970]. For the
purpose of this invention the sequence alignment was performed
using the publicly available software program MOE (Chemical
Computing Group Inc.) package version 2002.3, as described in the
accompanying User's Manual. When using the MOE program, alignment
was performed in the sequence editor window using the ALIGN option
utilizing the following program parameters: Initial pairwise
Build-up: ON, Substitution Matrix: Blosum62, Round Robin: ON, Gap
Start: 7, Gap Extend: 1, Iterative Refinement: ON, Build-up:
TREE-BASED, Secondary Structure: NONE, Structural Alignment:
ENABLED, Gap Start: 1, Gap Extend: 0.1
Once aligned, a rigid body fitting operation is performed where the
structure for the target protein is translated and rotated to
obtain an optimum fit relative to the structure of the reference
protein. The fitting operation uses an algorithm that computes the
optimum translation and rotation to be applied to the moving
structure, such that the root mean square deviation of the fit over
the specified pairs of equivalent atoms is an absolute minimum. For
the purpose of fitting operations made herein, the publicly
available software program MOE (Chemical Computing Group Inc.) v.
2002.3 was used.
The results from this process are typically reported as an RMSD
value between two sets of atoms. The term "root mean square
deviation" means the square root of the arithmetic mean of the
squares of deviations. It is a way to express the deviation or
variation from a trend or object. As used herein, an RMSD value
refers to a calculated value based on variations in the atomic
coordinates of a reference protein from the atomic coordinates of a
reference protein or portions of thereof. The structure coordinates
for MvaS, provided in FIG. 3, are used as the reference protein in
these calculations.
The same set of atoms was used for initial fitting of the
structures and for computing root mean square deviation values. For
example, if a root mean square deviation (RMSD) between C.alpha.
atoms of two proteins is needed, the proteins in question should be
superposed only on the C.alpha. atoms and not on any other set of
atoms. Similarly, if an RMSD calculation for all atoms is required,
the superposition of two structures should be performed on all
atoms.
Based on a review of protein structures deposited in the Protein
Databank (PDB), 1HND was identified as having the smallest RMSD
values relative to the structure coordinates provided herein. Table
7 below provides a series of RMSD values that were calculated by
the above described process using the structure coordinates in FIG.
3 as the reference protein and the structure coordinates from PDB
code: 1HND (3-oxoacyl-[acyl-carrier protein] synthase) as the
target protein.
TABLE-US-00007 TABLE 7 AA RESIDUES USED PORTION OF EACH AA TO
PERFORM RMSD RESIDUE USED TO PERFORM COMPARISON WITH RMSD
COMPARISON WITH RMSD PDB:1VR2 PDB:1HND [.ANG.] Table 2 alpha-carbon
atoms.sup.1 1.96 (4 Angstrom set) main-chain atoms.sup.1 1.82 all
non-hydrogen.sup.2 2.15 Table 3 alpha-carbon atoms.sup.1 1.78 (7
Angstrom set) main-chain atoms.sup.1 1.67 all non-hydrogen.sup.2
1.98 Table 4 alpha-carbon atoms.sup.1 1.96 (10 Angstrom set)
main-chain atoms.sup.1 1.91 all non-hydrogen.sup.2 2.23 2-384 of
alpha-carbon atoms.sup.1 2.60 SEQ. ID No. 1 main-chain atoms.sup.1
2.62 all non-hydrogen.sup.2 2.84 .sup.1the RMSD computed between
the atoms of all amino acids that are common to both the target and
the reference in the aligned and superposed structure. The amino
acids need not to be identical. .sup.2the RMSD computed only
between identical amino acids, which are common to both the target
and the reference in the aligned and superposed structure.
It is noted that mutants and variants of MvaS are likely to have
similar structures despite having different sequences. For example,
the binding pockets of these related proteins are likely to have
similar contours. Accordingly, it should be recognized that the
structure coordinates and binding pocket models provided herein
have utility for these other related proteins.
Accordingly, in one embodiment, the invention relates to data,
computer readable media comprising data, and uses of the data where
the data comprises all or a portion of the structure coordinates
shown in FIG. 3 or structure coordinates having a root mean square
deviation (RMSD) equal to or less than the RMSD value specified in
Columns 3, 4 or 5 of Table 1 when compared to the structure
coordinates of FIG. 3, the root mean square deviation being
calculated such that the portion of amino acid residues specified
in Column 2 of Table 1 of each set of structure coordinates are
superimposed and the root mean square deviation is based only on
those amino acid residues in the structure coordinates that are
also present in the portion of the protein specified in specified
in Column 1 of Table 1.
As noted, there are many different ways to express the surface
contours of the MvaS structure other than by using the structure
coordinates provided in FIG. 3. Accordingly, it is noted that the
present invention is also directed to any data, computer readable
media comprising data, and uses of the data where the data defines
a computer model for a protein binding pocket, at least a portion
of the computer model having a surface contour that has a root mean
square deviation equal to or less than a given RMSD value specified
in Columns 3, 4 or 5 of Table 1 when the coordinates used to
compute the surface contour are compared to the structure
coordinates of FIG. 3, wherein (a) the root mean square deviation
is calculated by the calculation method set forth herein, (b) the
portion of amino acid residues associated with the given RMSD value
in Table 1 (specified in Column 2 of Table 1) are superimposed
according to the RMSD calculation, and (c) the root mean square
deviation is calculated based only on those amino acid residues
present in both the protein being modeled and the portion of the
protein associated with the given RMSD in Table 1 (specified in
Column 1 of Table 1).
5. MvaS Structure
The present invention is also directed to a three-dimensional
crystal structure of MvaS. This crystal structure may be used to
identify binding sites, to provide mutants having desirable binding
properties, and ultimately, to design, characterize, or identify
ligands that interact with MvaS as well as other structurally
similar proteins.
The three-dimensional crystal structure of MvaS may be generated,
as is known in the art, from the structure coordinates shown in
FIG. 3 and similar such coordinates.
During the course of structure solution it became evident that the
crystals of MvaS of the present invention contained two MvaS
molecules in the asymmetric unit. It is noted that the sequence of
the structure coordinates presented in FIG. 3 differ in some
regards from the sequence shown in SEQ. ID No. 1. The final refined
coordinates do not include amino acid residues 1 and 384 391 in
molecule A and residues 1 and 388 391 in molecule B because the
electron density obtained was insufficient to identify their
position. The final coordinate set additionally includes 747
solvent molecules modeled as water and two molecules of
HMG-CoA.
FIG. 4 illustrates a ribbon diagram overview of the structure of
MvaS, highlighting the secondary structural elements of the
protein. As can be seen, the monomer exhibits a five-layered core
structure, .alpha.-.beta.-.alpha.-.beta.-.alpha., where each
.alpha. comprises two .alpha.-helices and each .beta. is made of a
five-stranded, mixed .beta.-sheet. The active site of MvaS is
occupied by its reaction product HMG-CoA, which binds in a deep
tunnel that is lined with conserved aromatic and hydrophobic
residues. Catalytic Cys111, which biochemical studies have
implicated as forming an enzyme-S-acetyl intermediate, is located
at the base of the tunnel and is unmodified in this product bound
structure.
The MvaS dimer is large and primarily hydrophobic with the
interface spanning several secondary structure region.
Complementary interactions between .alpha.5, .beta.6, and .alpha.6
link the catalytic site at Cys111 and form a continuous 10-stranded
.beta. sheet in the dimer. Additional interactions, including those
between the .beta.9-.alpha.7 loop of one monomer, with the
.alpha.5-.beta.5 loop and a 3-stranded antiparallel .beta.-sheet
containing domain of the other complete the dimer interface.
6. MvaS Active Site and Ligand Interaction
The term "binding site" or "binding pocket", as the terms are used
herein, refers to a region of a protein that, as a result of its
shape, favorably associates with a ligand or substrate. The term
"MvaS-like binding pocket" refers to a portion of a molecule or
molecular complex whose shape is sufficiently similar to the MvaS
binding pockets as to bind common ligands. This commonality of
shape may be quantitatively defined based on a comparison to a
reference point, that reference point being the structure
coordinates provided herein. For example the commonality of shape
may be quantitatively defined based on a root mean square deviation
(RMSD) from the structure coordinates of the backbone atoms of the
amino acids that make up the binding pockets in MvaS (as set forth
in FIG. 3).
The "active site binding pockets" or "active site" of MvaS refers
to the area on the surface of MvaS where the substrate binds.
FIG. 5 illustrates the HMG-CoA binding site of MvaS based on the
determined crystal structure for the molecule in the asymmetric
unit corresponding to the structure coordinates shown in FIG. 3.
The catalytic site for HMG-CoA is located in the long hydrophobic
tunnel constructed from conserved residues that emanate from
.alpha.7, as well as several loop regions. These regions include
residues from the .beta.9-.alpha.7 loop, the .beta.11-.beta.12
loop, the .beta.6-.alpha.6 loop, the .beta.5-.alpha.5 loop, the
.beta.7-.beta.8 loop, .alpha.10-.alpha.11 loop, and the
.beta.10-.alpha.9 loop (FIG. 4). All the residues within 4 .ANG. of
the HMG-CoA binding pocket as described in table 2 are illustrated
in FIG. 5.
In resolving the crystal structure of MvaS, applicants determined
that MvaS amino acids shown in Table 2 (above) are encompassed
within a 4-Angstrom radius around the MvaS active site and
therefore are likely close enough to interact with an active site
inhibitor of MvaS. Applicants have also determined that the amino
acids shown in Table 3 (above) are encompassed within a 7-Angstrom
radius around the MvaS active site. Further, the amino acids shown
in Table 4 (above) are encompassed within a 10-Angstrom radius
around the MvaS active site. Due to their proximity to the active
site, the amino acids in the 4, 7, and/or 10 Angstroms sets are
preferably conserved in variants of MvaS. While it is desirable to
largely conserve these residues, it should be recognized however
that variants may also involve varying 1, 2, 3, 4 or more of the
residues set forth in Tables 2, 3 and 4 in order to evaluate the
roles these amino acids play in the binding pocket.
With the knowledge of the MvaS crystal structure provided herein,
Applicants are able to know the contour of an MvaS binding pocket
based on the relative positioning of the 4, 7, and/or 10 Angstroms
sets of amino acids. Again, it is noted that it may be desirable to
form variants where 1, 2, 3, 4 or more of the residues set forth in
Tables 2, 3 and 4 are varied in order to evaluate the roles these
amino acids play in the binding pocket. Accordingly, any set of
structure coordinates for a protein from any source shall be
considered within the scope of the present invention if the
structure coordinates have a root mean square deviation equal to or
less than the RMSD value specified in Columns 3, 4 or 5 of Table 1
when compared to the structure coordinates of FIG. 3, the root mean
square deviation being calculated such that the portion of amino
acid residues specified in Column 2 of Table 1 of each set of
structure coordinates are superimposed and the root mean square
deviation is based only on those amino acid residues in the
structure coordinates that are also present in the portion of the
protein specified in specified in Column 1 of Table 1.
Accordingly, in various embodiments, the invention relates to data,
computer readable media comprising data, and uses of the data where
the data comprises structure coordinates that have a root mean
square deviation equal to or less than the RMSD value specified in
Columns 3, 4 or 5 of Table 1 when compared to the structure
coordinates of FIG. 3, the root mean square deviation being
calculated such that the portion of amino acid residues specified
in Column 2 of Table 1 of each set of structure coordinates are
superimposed and the root mean square deviation is based only on
those amino acid residues in the structure coordinates that are
also present in the portion of the protein specified in specified
in Column 1 of Table 1.
As noted above, there are many different ways to express the
surface contours of the MvaS structure other than by using the
structure coordinates provided in FIG. 3. Accordingly, it is noted
that the present invention is also directed to any data, computer
readable media comprising data, and uses of the data where the data
defines a computer model for a protein binding pocket, at least a
portion of the computer model having a surface contour that has a
root mean square deviation equal to or less than a given RMSD value
specified in Columns 3, 4 or 5 of Table 1 when the coordinates used
to compute the surface contour are compared to the structure
coordinates of FIG. 3, wherein (a) the root mean square deviation
is calculated by the calculation method set forth herein, (b) the
portion of amino acid residues associated with the given RMSD value
in Table 1 (specified in Column 2 of Table 1) are superimposed
according to the RMSD calculation, and (c) the root mean square
deviation is calculated based only on those amino acid residues
present in both the protein being modeled and the portion of the
protein associated with the given RMSD in Table 1 (specified in
Column 1 of Table 1).
It will be readily apparent to those of skill in the art that the
numbering of amino acids in other isoforms of MvaS may be different
than that set forth for MvaS. Corresponding amino acids in other
isoforms of MvaS are easily identified by visual inspection of the
amino acid sequences or by using commercially available homology
software programs, as further described below.
7. System for Displaying the Three Dimensional Structure of
MvaS
The present invention is also directed to machine-readable data
storage media having data storage material encoded with
machine-readable data that comprises structure coordinates for
MvaS. The present invention is also directed to a machine readable
data storage media having data storage material encoded with
machine readable data, which, when read by an appropriate machine,
can display a three dimensional representation of a structure of
MvaS.
All or a portion of the MvaS coordinate data shown in FIG. 3, when
used in conjunction with a computer programmed with software to
translate those coordinates into the three-dimensional structure of
MvaS may be used for a variety of purposes, especially for purposes
relating to drug discovery. Software for generating
three-dimensional graphical representations are known and
commercially available. The ready use of the coordinate data
requires that it be stored in a computer-readable format. Thus, in
accordance with the present invention, data capable of being
displayed as the three-dimensional structure of MvaS and/or
portions thereof and/or their structurally similar variants may be
stored in a machine-readable storage medium, which is capable of
displaying a graphical three-dimensional representation of the
structure.
For example, in various embodiments, a computer is provided for
producing a three-dimensional representation of at least an
MvaS-like binding pocket, the computer comprising:
machine readable data storage medium comprising a data storage
material encoded with machine-readable data, the machine readable
data comprising structure coordinates that have a root mean square
deviation equal to or less than the RMSD value specified in Columns
3, 4 or 5 of Table 1 when compared to the structure coordinates of
FIG. 3, the root mean square deviation being calculated such that
the portion of amino acid residues specified in Column 2 of Table 1
of each set of structure coordinates are superimposed and the root
mean square deviation is based only on those amino acid residues in
the structure coordinates that are also present in the portion of
the protein specified in specified in Column 1 of Table 1;
a working memory for storing instructions for processing the
machine-readable data;
a central-processing unit coupled to the working memory and to the
machine-readable data storage medium, for processing the
machine-readable data into the three-dimensional representation;
and
an output hardware coupled to the central processing unit, for
receiving the three dimensional representation.
Another embodiment of this invention provides a machine-readable
data storage medium, comprising a data storage material encoded
with machine readable data which, when used by a machine programmed
with instructions for using said data, displays a graphical
three-dimensional representation comprising MvaS or a portion or
variant thereof.
In various variations, the machine readable data comprises data for
representing a protein based on structure coordinates where the
structure coordinates have a root mean square deviation equal to or
less than the RMSD value specified in Columns 3, 4 or 5 of Table 1
when compared to the structure coordinates of FIG. 3, the root mean
square deviation being calculated such that the portion of amino
acid residues specified in Column 2 of Table 1 of each set of
structure coordinates are superimposed and the root mean square
deviation is based only on those amino acid residues in the
structure coordinates that are also present in the portion of the
protein specified in specified in Column 1 of Table 1.
According to another embodiment, the machine-readable data storage
medium comprises a data storage material encoded with a first set
of machine readable data which comprises the Fourier transform of
structure coordinates that have a root mean square deviation equal
to or less than the RMSD value specified in Columns 3, 4 or 5 of
Table 1 when compared to the structure coordinates of FIG. 3, the
root mean square deviation being calculated such that the portion
of amino acid residues specified in Column 2 of Table 1 of each set
of structure coordinates are superimposed and the root mean square
deviation is based only on those amino acid residues in the
structure coordinates that are also present in the portion of the
protein specified in specified in Column 1 of Table 1, and which,
when using a machine programmed with instructions for using said
data, can be combined with a second set of machine readable data
comprising the X-ray diffraction pattern of another molecule or
molecular complex to determine at least a portion of the structure
coordinates corresponding to the second set of machine readable
data. For example, the Fourier transform of the structure
coordinates set forth in FIG. 3 may be used to determine at least a
portion of the structure coordinates of other MvaS-like enzymes,
and isoforms of MvaS.
Optionally, a computer system is provided in combination with the
machine-readable data storage medium provided herein. In one
embodiment, the computer system comprises a working memory for
storing instructions for processing the machine-readable data; a
processing unit coupled to the working memory and to the
machine-readable data storage medium, for processing the
machine-readable data into the three-dimensional representation;
and an output hardware coupled to the processing unit, for
receiving the three-dimensional representation.
FIG. 6 illustrates an example of a computer system that may be used
in combination with storage media according to the present
invention. As illustrated, the computer system 10 includes a
computer 11 comprising a central processing unit ("CPU") 20, a
working memory 22 which may be, e.g., RAM (random-access memory) or
"core" memory, mass storage memory 24 (such as one or more disk
drives or CD-ROM drives), one or more cathode-ray tube ("CRT")
display terminals 26, one or more keyboards 28, one or more input
lines 30, and one or more output lines 40, all of which are
interconnected by a conventional bi-directional system bus 50.
Input hardware 36, coupled to computer 11 by input lines 30, may be
implemented in a variety of ways. For example, machine-readable
data of this invention may be inputted via the use of a modem or
modems 32 connected by a telephone line or dedicated data line 34.
Alternatively or additionally, the input hardware 36 may comprise
CD-ROM drives or disk drives 24. In conjunction with display
terminal 26, keyboard 28 may also be used as an input device.
Conventional devices may, similarly implement output hardware 46,
coupled to computer 11 by output lines 40. By way of example,
output hardware 46 may include CRT display terminal 26 for
displaying a graphical representation of a binding pocket of this
invention using a program such as MOE as described herein. Output
hardware might also include a printer 42, so that hard copy output
may be produced, or a disk drive 24, to store system output for
later use.
In operation, CPU 20 coordinates the use of the various input and
output devices 36, 46 coordinates data accesses from mass storage
24 and accesses to and from working memory 22, and determines the
sequence of data processing steps. A number of programs may be used
to process the machine-readable data of this invention. Such
programs are discussed in reference to using the three dimensional
structure of MvaS described herein.
The storage medium encoded with machine-readable data according to
the present invention can be any conventional data storage device
known in the art. For example, the storage medium can be a
conventional floppy diskette or hard disk. The storage medium can
also be an optically readable data storage medium, such as a CD-ROM
or a DVD-ROM, or a rewritable medium such as a magneto-optical disk
that is optically readable and magneto-optically writable.
8. Uses of the Three Dimensional Structure of MvaS
The three-dimensional crystal structure of the present invention
may be used to identify MvaS binding sites, be used as a molecular
replacement model to solve the structure of unknown crystallized
proteins, to design mutants having desirable binding properties,
and ultimately, to design, characterize, identify entities capable
of interacting with MvaS and other structurally similar proteins as
well as other uses that would be recognized by one of ordinary
skill in the art. Such entities may be chemical entities or
proteins. The term "chemical entity", as used herein, refers to
chemical compounds, complexes of at least two chemical compounds,
and fragments of such compounds.
The MvaS structure coordinates provided herein are useful for
screening and identifying drugs that inhibit MvaS and other
structurally similar proteins. For example, the structure encoded
by the data may be computationally evaluated for its ability to
associate with putative substrates or ligands. Such compounds that
associate with MvaS may inhibit MvaS, and are potential drug
candidates. Additionally or alternatively, the structure encoded by
the data may be displayed in a graphical three-dimensional
representation on a computer screen. This allows visual inspection
of the structure, as well as visual inspection of the structure's
association with the compounds.
Thus, according to another embodiment of the present invention, a
method is provided for evaluating the potential of an entity to
associate with MvaS or a fragment or variant thereof by using all
or a portion of the structure coordinates provided in FIG. 3 or
functional equivalents thereof. A method is also provided for
evaluating the potential of an entity to associate with MvaS or a
fragment or variant thereof by using structure coordinates similar
to all or a portion of the structure coordinates provided in FIG. 3
or functional equivalents thereof.
The method may optionally comprise the steps of: creating a
computer model of all or a portion of a protein structure (e.g., a
binding pocket) using structure coordinates according to the
present invention; performing a fitting operation between the
entity and the computer model; and analyzing the results of the
fitting operation to quantify the association between the entity
and the model. The portion of the protein structure used optionally
comprises all of the amino acids listed in Tables 2, 3 and 4 that
are present in the structure coordinates being used.
It is noted that the computer model may not necessarily directly
use the structure coordinates. Rather, a computer model can be
formed that defines a surface contour that is the same or similar
to the surface contour defined by the structure coordinates.
The structure coordinates provided herein can also be utilized in a
method for identifying a ligand (e.g., entities capable of
associating with a protein) of a protein comprising an MvaS-like
binding pocket. One embodiment of the method comprises: using all
or a portion of the structure coordinates provided herein to
generate a three-dimensional structure of an MvaS-like binding
pocket; employing the three-dimensional structure to design or
select a potential ligand; synthesizing the potential ligand; and
contacting the synthesized potential ligand with a protein
comprising an MvaS-like binding pocket to determine the ability of
the potential ligand to interact with protein. According to this
method, the structure coordinates used may have a root mean square
deviation equal to or less than the RMSD values specified in
Columns 3, 4 or 5 of Table 1 when compared to the structure
coordinates of FIG. 3 according to the RMSD calculation method set
forth herein, provided that the portion of amino acid residues
specified in Column 2 of Table 1 of each set of structure
coordinates are superimposed and the root mean square deviation is
calculated based only on those amino acid residues in the structure
coordinates that are also present in the portion of the protein
specified in Column 1 of Table 1. The portion of the protein
structure used optionally comprises all of the amino acids listed
in Tables 2, 3, and/or 4 that are present.
As noted previously, the three-dimensional structure of an
MvaS-like binding pocket need not be generated directly from
structure coordinates. Rather, a computer model can be formed that
defines a surface contour that is the same or similar to the
surface contour defined by the structure coordinates.
A method is also provided for evaluating the ability of an entity,
such as a compound or a protein to associate with an MvaS-like
binding pocket, the method comprising: constructing a computer
model of a binding pocket defined by structure coordinates that
have a root mean square deviation equal to or less than the RMSD
value specified in Columns 3, 4 or 5 of Table 1 when compared to
the structure coordinates of FIG. 3, the root mean square deviation
being calculated such that the portion of amino acid residues
specified in Column 2 of Table 1 of each set of structure
coordinates are superimposed and the root mean square deviation is
based only on those amino acid residues in the structure
coordinates that are also present in the portion of the protein
specified in specified in Column 1 of Table 1; selecting an entity
to be evaluated by a method selected from the group consisting of
(i) assembling molecular fragments into the entity, (ii) selecting
an entity from a small molecule database, (iii) de novo ligand
design of the entity, and (iv) modifying a known ligand for MvaS,
or a portion thereof; performing a fitting program operation
between computer models of the entity to be evaluated and the
binding pocket in order to provide an energy-minimized
configuration of the entity in the binding pocket; and evaluating
the results of the fitting operation to quantify the association
between the entity and the binding pocket model in order to
evaluate the ability of the entity to associate with the said
binding pocket.
The computer model of a binding pocket used in this embodiment need
not be generated directly from structure coordinates. Rather, a
computer model can be formed that defines a surface contour that is
the same or similar to the surface contour defined by the structure
coordinates.
Also according to the method, the method may further include
synthesizing the entity; and contacting a protein having an
MvaS-like binding pocket with the synthesized entity.
With the structure provided herein, the present invention for the
first time permits the use of molecular design techniques to
identify, select or design potential inhibitors of MvaS, based on
the structure of an MvaS-like binding pocket. Such a predictive
model is valuable in light of the high costs associated with the
preparation and testing of the many diverse compounds that may
possibly bind to the MvaS protein.
According to this invention, a potential MvaS inhibitor may now be
evaluated for its ability to bind an MvaS-like binding pocket prior
to its actual synthesis and testing. If a proposed entity is
predicted to have insufficient interaction or association with the
binding pocket, preparation and testing of the entity can be
obviated. However, if the computer modeling indicates a strong
interaction, the entity may then be obtained and tested for its
ability to bind.
A potential inhibitor of an MvaS-like binding pocket may be
computationally evaluated using a series of steps in which chemical
entities or fragments are screened and selected for their ability
to associate with the MvaS-like binding pockets.
One skilled in the art may use one of several methods to screen
entities (whether chemical or protein) for their ability to
associate with an MvaS-like binding pocket. This process may begin
by visual inspection of, for example, an MvaS-like binding pocket
on a computer screen based on the MvaS structure coordinates in
FIG. 3 or other coordinates which define a similar shape generated
from the machine-readable storage medium. Selected fragments or
chemical entities may then be positioned in a variety of
orientations, or docked, within that binding pocket as defined
above. Docking may be accomplished using software such as Quanta
and Sybyl, followed by energy minimization and molecular dynamics
with standard molecular mechanics force fields, such as CHARMM and
AMBER.
Specialized computer programs may also assist in the process of
selecting entities. These include: GRID (P. J. Goodford, "A
Computational Procedure for Determining Energetically Favorable
Binding Sites on Biologically Important Macromolecules", J. Med.
Chem., 28, pp. 849 857 (1985)). GRID is available from Oxford
University, Oxford, UK; MCSS (A. Miranker et al., "Functionality
Maps of Binding Sites: A Multiple Copy Simultaneous Search Method."
Proteins: Structure, Function and Genetics, 11, pp. 29 34 (1991)).
MCSS is available from Molecular Simulations, San Diego, Calif.;
AUTODOCK (D. S. Goodsell et al., "Automated Docking of Substrates
to Proteins by Simulated Annealing", Proteins: Structure, Function,
and Genetics, 8, pp. 195 202 (1990)). AUTODOCK is available from
Scripps Research Institute, La Jolla, Calif.; & DOCK (I. D.
Kuntz et al., "A Geometric Approach to Macromolecule-Ligand
Interactions", J. Mol. Biol., 161, pp. 269 288 (1982)). DOCK is
available from University of California, San Francisco, Calif.
Once suitable entities have been selected, they can be designed or
assembled. Assembly may be preceded by visual inspection of the
relationship of the fragments to each other on the
three-dimensional image displayed on a computer screen in relation
to the structure coordinates of MvaS. This may then be followed by
manual model building using software such as MOE, QUANTA or Sybyl
[Tripos Associates, St. Louis, Mo].
Useful programs to aid one of skill in the art in connecting the
individual chemical entities or fragments include: CAVEAT (P. A.
Bartlett et al, "CAVEAT: A Program to Facilitate the
Structure-Derived Design of Biologically Active Molecules", in
"Molecular Recognition in Chemical and Biological Problems",
Special Pub., Royal Chem. Soc., 78, pp. 182 196 (1989); G. Lauri
and P. A. Bartlett, "CAVEAT: a Program to Facilitate the Design of
Organic Molecules", J. Comput. Aided Mol. Des., 8, pp. 51 66
(1994)). CAVEAT is available from the University of California,
Berkeley, Calif.; 3D Database systems such as ISIS (MDL Information
Systems, San Leandro, Calif.). This area is reviewed in Y. C.
Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35,
pp. 2145 2154 (1992); HOOK (M. B. Eisen et al, "HOOK: A Program for
Finding Novel Molecular Architectures that Satisfy the Chemical and
Steric Requirements of a Macromolecule Binding Site", Proteins:
Struct., Funct., Genet., 19, pp. 199 221 (1994). HOOK is available
from Molecular Simulations, San Diego, Calif.
Instead of proceeding to build an inhibitor of an MvaS-like binding
pocket in a step-wise fashion one fragment or entity at a time as
described above, inhibitory or other MvaS binding compounds may be
designed as a whole or "de novo" using either an empty binding site
or optionally including some portion(s) of a known inhibitor(s).
There are many de novo ligand design methods including: LUDI (H.-J.
Bohm, "The Computer Program LUDI: A New Method for the De Novo
Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp.
61 78 (1992)). LUDI is available from Molecular Simulations
Incorporated, San Diego, Calif.; LEGEND (Y. Nishibata et al.,
Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from
Molecular Simulations Incorporated, San Diego, Calif.; LEAPFROG
(available from Tripos Associates, St. Louis, Mo.); & SPROUT
(V. Gillet et al, "SPROUT: A Program for Structure Generation)", J.
Comput. Aided Mol. Design, 7, pp. 127 153 (1993)). SPROUT is
available from the University of Leeds, UK.
Other molecular modeling techniques may also be employed in
accordance with this invention (see, e.g., Cohen et al., "Molecular
Modeling Software and Methods for Medicinal Chemistry, J. Med.
Chem., 33, pp. 883 894 (1990); see also, M. A. Navia and M. A.
Murcko, "The Use of Structural Information in Drug Design", Current
Opinions in Structural Biology, 2, pp. 202 210 (1992); L. M. Balbes
et al., "A Perspective of Modern Methods in Computer-Aided Drug
Design", in Reviews in Computational Chemistry, Vol. 5, K. B.
Lipkowitz and D. B. Boyd, Eds., VCH, New York, pp. 337 380 (1994);
see also, W. C. Guida, "Software For Structure-Based Drug Design",
Curr. Opin. Struct. Biology, 4, pp. 777 781 (1994)).
Once an entity has been designed or selected, for example, by the
above methods, the efficiency with which that entity may bind to an
MvaS binding pocket may be tested and optimized by computational
evaluation. For example, an effective MvaS binding pocket inhibitor
preferably demonstrates a relatively small difference in energy
between its bound and free states (i.e., a small deformation energy
of binding). Thus, the most efficient MvaS binding pocket
inhibitors should preferably be designed with deformation energy of
binding of not greater than about 10 kcal/mole, more preferably,
not greater than 7 kcal/mole. MvaS binding pocket inhibitors may
interact with the binding pocket in more than one of multiple
conformations that are similar in overall binding energy. In those
cases, the deformation energy of binding is taken to be the
difference between the energy of the free entity and the average
energy of the conformations observed when the inhibitor binds to
the protein.
An entity designed or selected as binding to an MvaS binding pocket
may be further computationally optimized so that in its bound state
it would preferably lack repulsive electrostatic interaction with
the target enzyme and with the surrounding water molecules. Such
non-complementary electrostatic interactions include repulsive
charge--charge, dipole--dipole and charge-dipole interactions.
Specific computer software is available in the art to evaluate
compound deformation energy and electrostatic interactions.
Examples of programs designed for such uses include: Gaussian 94,
revision C (M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa. COPYRGT.
1995); AMBER, version 4.1 (P. A. Kollman, University of California
at San Francisco, COPYRGT 1995); QUANTA/CHARMM (Molecular
Simulations, Inc., San Diego, Calif. COPYRGT. 1995); Insight
II/Discover (Molecular Simulations, Inc., San Diego, Calif.
COPYRGT. 1995); DelPhi (Molecular Simulations, Inc., San Diego,
Calif. COPYRGT. 1995); and AMSOL (Quantum Chemistry Program
Exchange, Indiana University). These programs may be implemented,
for instance, using a Silicon Graphics workstation such as an
Indigo.sup.2 with "IMPACT" graphics. Other hardware systems and
software packages will be known to those skilled in the art.
Another approach provided by this invention, is the computational
screening of small molecule databases for chemical entities or
compounds that can bind in whole, or in part, to an MvaS binding
pocket. In this screening, the quality of fit of such entities to
the binding site may be judged either by shape complementarities or
by estimated interaction energy [E. C. Meng et al., J. Comp. Chem.,
13, 505 524 (1992)].
According to another embodiment, the invention provides compounds
that associate with an MvaS-like binding pocket produced or
identified by various methods set forth above.
The structure coordinates set forth in FIG. 3 can also be used to
aid in obtaining structural information about another crystallized
molecule or molecular complex. This may be achieved by any of a
number of well-known techniques, including molecular
replacement.
For example, a method is also provided for utilizing molecular
replacement to obtain structural information about a protein whose
structure is unknown comprising the steps of: generating an X-ray
diffraction pattern of a crystal of the protein whose structure is
unknown; generating a three-dimensional electron density map of the
protein whose structure is unknown from the X-ray diffraction
pattern by using at least a portion of the structure coordinates
set forth in FIG. 3 as a molecular replacement model.
By using molecular replacement, all or part of the structure
coordinates of the MvaS provided by this invention (and set forth
in FIG. 3) can be used to determine the structure of another
crystallized molecule or molecular complex more quickly and
efficiently than attempting an ab initio structure determination.
One particular use includes use with other structurally similar
proteins. Molecular replacement provides an accurate estimation of
the phases for an unknown structure. Phases are a factor in
equations used to solve crystal structures that cannot be
determined directly. Obtaining accurate values for the phases, by
methods other than molecular replacement, is a time-consuming
process that involves iterative cycles of approximations and
refinements and greatly hinders the solution of crystal structures.
However, when the crystal structure of a protein containing at
least a homologous portion has been solved, the phases from the
known structure provide a satisfactory estimate of the phases for
the unknown structure.
Thus, this method involves generating a preliminary model of a
molecule or molecular complex whose structure coordinates are
unknown, by orienting and positioning the relevant portion of MvaS
according to FIG. 3 within the unit cell of the crystal of the
unknown molecule or molecular complex so as best to account for the
observed X-ray diffraction pattern of the crystal of the molecule
or molecular complex whose structure is unknown. Phases can then be
calculated from this model and combined with the observed X-ray
diffraction pattern amplitudes to generate an electron density map
of the structure whose coordinates are unknown. This, in turn, can
be subjected to any well-known model building and structure
refinement techniques to provide a final, accurate structure of the
unknown crystallized molecule or molecular complex [E. Lattman,
"Use of the Rotation and Translation Functions", in Meth. Enzymol.,
115, pp. 55 77 (1985); M. G. Rossmann, ed., "The Molecular
Replacement Method", Int. Sci. Rev. Ser., No. 13, Gordon &
Breach, New York (1972)].
The structure of any portion of any crystallized molecule or
molecular complex that is sufficiently homologous to any portion of
MvaS can be resolved by this method.
In one embodiment, the method of molecular replacement is utilized
to obtain structural information about the present invention and
any other MvaS-like molecule. The structure coordinates of MvaS, as
provided by this invention, are particularly useful in solving the
structure of other isoforms of MvaS or MvaS complexes.
The structure coordinates of MvaS as provided by this invention are
useful in solving the structure of MvaS variants that have amino
acid substitutions, additions and/or deletions (referred to
collectively as "MvaS mutants", as compared to naturally occurring
MvaS). These MvaS mutants may optionally be crystallized in
co-complex with a ligand, such as an inhibitor, substrate analogue
or a suicide substrate. The crystal structures of a series of such
complexes may then be solved by molecular replacement and compared
with that of MvaS. Potential sites for modification within the
various binding sites of the enzyme may thus be identified. This
information provides an additional tool for determining the most
efficient binding interactions such as, for example, increased
hydrophobic interactions, between MvaS and a ligand. It is noted
that the ligand may be the protein's natural ligand or may be a
potential agonist or antagonist of a protein.
All of the complexes referred to above may be studied using
well-known X-ray diffraction techniques and may be refined versus
1.5 3 .ANG. resolution X-ray data to an R value of about 0.22 or
less using computer software, such as X-PLOR [Yale University,
COPYRIGHT. 1992, distributed by Molecular Simulations, Inc.; see,
e.g., Blundell & Johnson, supra; Meth. Enzymol., Vol. 114 &
115, H. W. Wyckoff et al., eds., Academic Press (1985)]. This
information may thus be used to optimize known MvaS inhibitors, and
more importantly, to design new MvaS inhibitors.
The structure coordinates described above may also be used to
derive the dihedral angles, phi and psi, that define the
conformation of the amino acids in the protein backbone. As will be
understood by those skilled in the art, the phi.sub.n angle refers
to the rotation around the bond between the alpha-carbon and the
nitrogen, and the psi.sub.n angle refers to the rotation around the
bond between the carbonyl carbon and the alpha-carbon. The
subscript "n" identifies the amino acid whose conformation is being
described [for a general reference, see Blundell and Johnson,
Protein Crystallography, Academic Press, London, 1976].
9. Uses of the Crystal and Diffraction Pattern of MvaS
Crystals, crystallization conditions and the diffraction pattern of
MvaS that can be generated from the crystals also have a range of
uses. One particular use relates to screening entities that are not
known ligands of MvaS for their ability to bind to MvaS. For
example, with the availability of crystallization conditions,
crystals and diffraction patterns of MvaS provided according to the
present invention, it is possible to take a crystal of MvaS; expose
the crystal to one or more entities that may be a ligand of MvaS;
and determine whether a ligand/MvaS complex is formed. The crystals
of MvaS may be exposed to potential ligands by various methods,
including but not limited to, soaking a crystal in a solution of
one or more potential ligands or co-crystallizing MvaS in the
presence of one or more potential ligands. Given the structure
coordinates provided herein, once a ligand complex is formed, the
structure coordinates can be used as a model in molecular
replacement in order to determine the structure of the ligand
complex.
Once one or more ligands are identified, structural information
from the ligand/MvaS complex(es) may be used to design new ligands
that bind tighter, bind more specifically, have better biological
activity or have better safety profile than known ligands.
In one embodiment, a method is provided for identifying a ligand
that binds to MvaS comprising: (a) attempting to crystallize a
protein that comprises a sequence wherein at least a portion of the
sequence has 55%, 65%, 75%, 85%, 90%, 95%, 97%, 99% or greater
identity with SEQ. ID No. 1 in the presence of one or more
entities; (b) if crystals of the protein are obtained in step (a),
obtaining an X-ray diffraction pattern of the protein crystal; and
(c) determining whether a ligand/protein complex was formed by
comparing an X-ray diffraction pattern of a crystal of the protein
formed in the absence of the one or more entities to the crystal
formed in the presence of the one or more entities.
In another embodiment, a method is provided for identifying a
ligand that binds to MvaS comprising: soaking a crystal of a
protein wherein at least a portion of the protein has 55%, 65%,
75%, 85%, 90%, 95%, 97%, 99% or greater identity with SEQ. ID No. 1
with one or more entities; determining whether a ligand/protein
complex was formed by comparing an X-ray diffraction pattern of a
crystal of the protein that has not been soaked with the one or
more entities to the crystal that has been soaked with the one or
more entities.
Optionally, the method may further comprise converting the
diffraction patterns into electron density maps using phases of the
protein crystal and comparing the electron density maps.
Libraries of "shape-diverse" compounds may optionally be used to
allow direct identification of the ligand-receptor complex even
when the ligand is exposed as part of a mixture. According to this
variation, the need for time-consuming de-convolution of a hit from
the mixture is avoided. More specifically, the calculated electron
density function reveals the binding event, identifies the bound
compound and provides a detailed 3-D structure of the
ligand-receptor complex. Once a hit is found, one may optionally
also screen a number of analogs or derivatives of the hit for
tighter binding or better biological activity by traditional
screening methods. The hit and information about the structure of
the target may also be used to develop analogs or derivatives with
tighter binding or better biological activity. It is noted that the
ligand-MvaS complex may optionally be exposed to additional
iterations of potential ligands so that two or more hits can be
linked together to make a more potent ligand. Screening for
potential ligands by co-crystallization and/or soaking is further
described in U.S. Pat. No. 6,297,021, which is incorporated herein
by reference.
EXAMPLES
Example 1
Expression and Purification of MvaS Ef
This example describes the expression of MvaS_Ef. It should be
noted that a variety of other expression systems and hosts are also
suitable for the expression of MvaS_Ef, as would be readily
appreciated by one of skill in the art.
The gene encoding residues 1 383 (from SEQ. ID No. 1), which
corresponds to the full-length MvaS from E. faecalis, was isolated
by PCR from E. faecalis genomic DNA (ATCC700800D) and cloned into
the TOPO-activated cloning site of pSX26 vector. This DNA sequence
is presented in SEQ. ID No. 2. Expression in this vector generated
a fusion of the full-length MvaS with non-cleavable
carboxy-terminal six histidine tag, the amino acid sequence of
which is shown, underlined, in FIG. 1 (SEQ. ID No. 1). For
production of seleno methionine labeled protein, the expression
plasmid encoding for MvaS_Ef fused with carboxy-terminal histidine
tag was transformed into methionine auxotroph DL41 (Hendrickson, W.
1990, EMBO J. 9:1655 0).
Biomass for purification of recombinant seleno-methionine labeled
MvaS_Ef was generated using minimal media supplemented with
seleno-methionine (Sigma, MO) using 96-well fermentor. It should be
noted that a variety of other protocols and expression strains are
also suitable for the expression of selenomethionine derivative of
MvaS website:
cbr.med.harvard.edu/investigators/springer/lab/protocols/sara_SeMet
Doublie, S. (1997). Methods in Enzymology 276, 523 530; website:
novagen.com/docs/ndis/INNO10005.pdf as would be readily appreciated
by one of skill in the art. Cells from a single 70 ml fermentor
tubes was thawed by addition of 21 ml of lysis buffer (50 mM
Tris/HCl pH 7.9, 50 mM NaCl, 1 mM MgCl.sub.2) containing hen egg
white lysozyme (0.6 mg/ml) and Benzonase (2.5 U/ml) and sonicated
using Sonic Hedgehog robot. The sonicate was allowed to stand for
30 minutes at .about.4.degree. C. Total lysate was clarified by
centrifugation and 2 mL of 5M NaCl were added to the cleared
lysate. The cleared lysate from four fermentor tubes was applied to
3 ml bed ProBond column that had been equilibrated to 50 mM
Potassium Phosphate pH 7.8, 0.4 M NaCl, 0.1 M KCl, 20 mM imidazole,
10% glycerol, 0.25 mM TCEP. The solution was passed through the
column using gravity flow and the column was washed with 6 bed
volumes of 50 mM Potassium Phosphate pH 7.8, 0.4 M NaCl, 0.1 M KCl,
40 mM imidazole, 10% glycerol, 0.25 mM TCEP. The product was eluted
with 12 ml of 50 mM Potassium Phosphate pH 7.4, 0.4 M NaCl, 0.1 M
KCl, 200 mM imidazole, 10% glycerol, 0.25 mM TCEP. The eluted
protein was concentrated and buffer-exchanged into 25 mM Tris pH
7.9, 150 mM NaCl by using Vivaspin centrifugal concentrators.
Following three five-fold dilution buffer-exchanges, the IMAC
purified MvaS_Ef was concentrated to 10 mg/ml with a total volume
of 0.47 ml. The molecular weight of the purified protein
corresponded to the 100% incorporation of seleno-methionine as
determined by Mass Spectrograph (MS) analysis (43,443 observed and
43,436 expected without N-terminal methionine). Purified MvaS_Ef
exhibited a major band by both isoelectric focusing (IEF) and by
sodium-dodecyl-sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) analyses.
Example 2
Crystallization of MvaS
This example describes the crystallization of MvaS. It is noted
that the precise crystallization conditions used may be further
varied, for example by performing a fine screen based on these
crystallization conditions.
MvaS protein samples (corresponding to SEQ. ID No. 1) were
incubated with 5 mM HMG-CoA and 5 mM MgCl.sub.2 before setting
crystallization trials. Crystals were obtained after an extensive
and broad screen of conditions, followed by optimization.
Diffraction quality crystals were grown in 100 nL sitting droplets
using the vapor diffusion method. 50 nL comprising the MvaS-HMG-CoA
complex (10 mg/ml) was mixed with 50 nL from a reservoir solution
(100 .mu.L) comprising: 24% PEG MME 2000; and 0.1M Citrate buffer
pH-5.0. The resulting solution was incubated over a period of two
weeks at 4.degree. C. These crystals could also be obtained from
PEG MME 550, PEG 3350, PEG 4000 and PEG 6000, PEG 800 at 4.degree.
C. and 20.degree. C. Crystals typically appeared after 1 days and
grew to a maximum size within 3 7 days. Single crystals were
transferred, briefly, into a cryoprotecting solution containing the
reservoir solution supplemented with 25% v/v ethylene glycol.
Crystals were then flash frozen by immersion in liquid nitrogen and
then stored under liquid nitrogen. A crystal of the MvaS-HMG-CoA
complex produced as described is illustrated in FIG. 2.
While the present invention is disclosed with reference to certain
embodiments and examples detailed above, it is to be understood
that these embodiments and examples are intended to be illustrative
rather than limiting, as it is contemplated that modifications will
readily occur to those skilled in the art, which modifications are
intended to be within the scope of the invention and the appended
claims. All patents, papers, and books cited in this application
are incorporated herein in their entirety.
SEQUENCE LISTINGS
1
21391PRTEnterococcus faecalisFull-length E. faecalis MvaS(1)..(391)
1Met Thr Ile Gly Ile Asp Lys Ile Ser Phe Phe Val Pro Pro Tyr Tyr1 5
10 15Ile Asp Met Thr Ala Leu Ala Glu Ala Arg Asn Val Asp Pro Gly
Lys 20 25 30Phe His Ile Gly Ile Gly Gln Asp Gln Met Ala Val Asn Pro
Ile Ser 35 40 45Gln Asp Ile Val Thr Phe Ala Ala Asn Ala Ala Glu Ala
Ile Leu Thr 50 55 60Lys Glu Asp Lys Glu Ala Ile Asp Met Val Ile Val
Gly Thr Glu Ser65 70 75 80Ser Ile Asp Glu Ser Lys Ala Ala Ala Val
Val Leu His Arg Leu Met 85 90 95Gly Ile Gln Pro Phe Ala Arg Ser Phe
Glu Ile Lys Glu Ala Cys Tyr 100 105 110Gly Ala Thr Ala Gly Leu Gln
Leu Ala Lys Asn His Val Ala Leu His 115 120 125Pro Asp Lys Lys Val
Leu Val Val Ala Ala Asp Ile Ala Lys Tyr Gly 130 135 140Leu Asn Ser
Gly Gly Glu Pro Thr Gln Gly Ala Gly Ala Val Ala Met145 150 155
160Leu Val Ala Ser Glu Pro Arg Ile Leu Ala Leu Lys Glu Asp Asn Val
165 170 175Met Leu Thr Gln Asp Ile Tyr Asp Phe Trp Arg Pro Thr Gly
His Pro 180 185 190Tyr Pro Met Val Asp Gly Pro Leu Ser Asn Glu Thr
Tyr Ile Gln Ser 195 200 205Phe Ala Gln Val Trp Asp Glu His Lys Lys
Arg Thr Gly Leu Asp Phe 210 215 220Ala Asp Tyr Asp Ala Leu Ala Phe
His Ile Pro Tyr Thr Lys Met Gly225 230 235 240Lys Lys Ala Leu Leu
Ala Lys Ile Ser Asp Gln Thr Glu Ala Glu Gln 245 250 255Glu Arg Ile
Leu Ala Arg Tyr Glu Glu Ser Ile Val Tyr Ser Arg Arg 260 265 270Val
Gly Asn Leu Tyr Thr Gly Ser Leu Tyr Leu Gly Leu Ile Ser Leu 275 280
285Leu Glu Asn Ala Thr Thr Leu Thr Ala Gly Asn Gln Ile Gly Leu Phe
290 295 300Ser Tyr Gly Ser Gly Ala Val Ala Glu Phe Phe Thr Gly Glu
Leu Val305 310 315 320Ala Gly Tyr Gln Asn His Leu Gln Lys Glu Thr
His Leu Ala Leu Leu 325 330 335Asp Asn Arg Thr Glu Leu Ser Ile Ala
Glu Tyr Glu Ala Met Phe Ala 340 345 350Glu Thr Leu Asp Thr Asp Ile
Asp Gln Thr Leu Glu Asp Glu Leu Lys 355 360 365Tyr Ser Ile Ser Ala
Ile Asn Asn Thr Val Arg Ser Tyr Arg Asn Lys 370 375 380Gly His His
His His His His385 39021176DNAEnterococcus faecaliscDNA sequence
encoding MvaS(1)..(1175) 2atgacaattg ggattgataa aattagtttt
tttgtgcccc cttattatat tgatatgacg 60gcactggctg aagccagaaa tgtagaccct
ggaaaatttc atattggtat tgggcaagac 120caaatggcgg tgaacccaat
cagccaagat attgtgacat ttgcagccaa tgccgcagaa 180gcgatcttga
ccaaagaaga taaagaggcc attgatatgg tgattgtcgg gactgagtcc
240agtatcgatg agtcaaaagc ggccgcagtt gtcttacatc gtttaatggg
gattcaacct 300ttcgctcgct ctttcgaaat caaggaagct tgttacggag
caacagcagg cttacagtta 360gctaagaatc acgtagcctt acatccagat
aaaaaagtct tggtcgtagc ggcagatatt 420gcaaaatatg gcttaaattc
tggcggtgag cctacacaag gagctggggc ggttgcaatg 480ttagttgcta
gtgaaccgcg cattttggct ttaaaagagg ataatgtgat gctgacgcaa
540gatatctatg acttttggcg tccaacaggc cacccgtatc ctatggtcga
tggtcctttg 600tcaaacgaaa cctacatcca atcttttgcc caagtctggg
atgaacataa aaaacgaacc 660ggtcttgatt ttgcagatta tgatgcttta
gcgttccata ttccttacac aaaaatgggc 720aaaaaagcct tattagcaaa
aatctccgac caaactgaag cagaacagga acgaatttta 780gcccgttatg
aagaaagtat cgtctatagt cgtcgcgtag gaaacttgta tacgggttca
840ctttatctgg gactcatttc ccttttagaa aatgcaacga ctttaaccgc
aggcaatcaa 900attggtttat tcagttatgg ttctggtgct gtcgctgaat
ttttcactgg tgaattagta 960gctggttatc aaaatcattt acaaaaagaa
actcatttag cactgctgga taatcggaca 1020gaactttcta tcgctgaata
tgaagccatg tttgcagaaa ctttagacac agacattgat 1080caaacgttag
aagatgaatt aaaatatagt atttctgcta ttaataatac cgttcgttct
1140tatcgaaaca aagggcacca ccaccaccac cactag 1176
* * * * *